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Facilitation of lin-12-mediated signalling by sel-12, a Caenorhabditis elegans S182 Alzheimer's disease gene
Abstract
The lin-12 and glp-1 genes of Caenorhabditis elegans are members of the lin-12/Notch family of receptors for intercellular signals that specify cell fate. By screening for suppressors of a lin-12 gain-of-function mutation, we identified a new gene, sel-12, which appears to function in receiving cells to facilitate signalling mediated by lin-12 and glp-1. The sel-12 gene encodes a protein with multiple transmembrane domains, and is similar to S182, which has been implicated in early-onset familial Alzheimer's disease. The high degree of sequence conservation suggests that the function of the SEL-12 and S182 proteins may also be conserved.
... We introduced this transgene into three sel-12 mutants, sel-12(ar131), sel-12(ok2078), and sel-12(ty11). sel-12(ar131) mutants carry a missense mutation in the sel-12 gene that changes a cysteine to a tyrosine (C60Y), which is a conserved change observed in human presenilin that is associated with FAD (Levitan & Greenwald, 1995), sel-12(ok2078) mutants have a large deletion of the sel-12 locus and sel-12(ty11) mutants contain a premature stop codon in the sel-12 open reading frame (Cinar, Sweet, Hosemann, Earley, & Newman, 2001) (Figure 1a). From the analysis of the sel-12 mutants as day 1 adults, we found that they resemble wild-type animals showing an even distribution of Q35::YFP ( Figure 1b). ...
... Thus, to test the role of SEL-12's protease activity in proteostasis, using CRISPR/Cas9 technology, we introduced a knock-in D to A mutation in a residue critical (D226A) for aspartyl protease function (Sarasija et al., 2018;Sarasija & Norman, 2015) in the endogenous sel-12 gene product ( Figure 1a). First, like the sel-12 loss of function mutations, sel-12(ar131), sel-12(ok2078), and sel-12(ty11), sel-12(D->A) mutants show a severe egg-laying defect and protruding vulva consistent with loss of Notch signaling (Cinar et al., 2001;Levitan & Greenwald, 1995;Sarasija et al., 2018;Sarasija & Norman, 2015). Concordant with our previous studies, we found that the sel-12(D->A) mutants did not show signs of neurodegeneration or mitochondria defects. ...
Aging and age‐related diseases are associated with a decline of protein homeostasis (proteostasis), but the mechanisms underlying this decline are not clear. In particular, decreased proteostasis is a widespread molecular feature of neurodegenerative diseases, such as Alzheimer's disease (AD). Familial AD is largely caused by mutations in the presenilin encoding genes; however, their role in AD is not understood. In this study, we investigate the role of presenilins in proteostasis using the model system Caenorhabditis elegans. Previously, we found that mutations in C. elegans presenilin cause elevated ER to mitochondria calcium signaling, which leads to an increase in mitochondrial generated oxidative stress. This, in turn, promotes neurodegeneration. To understand the cellular mechanisms driving neurodegeneration, using several molecular readouts of protein stability in C. elegans, we find that presenilin mutants have widespread defects in proteostasis. Markedly, we demonstrate that these defects are independent of the protease activity of presenilin and that reduction in ER to mitochondrial calcium signaling can significantly prevent the proteostasis defects observed in presenilin mutants. Furthermore, we show that supplementing presenilin mutants with antioxidants suppresses the proteostasis defects. Our findings indicate that defective ER to mitochondria calcium signaling promotes proteostatic collapse in presenilin mutants by increasing oxidative stress. Ashkavand et al uncover a critical role of ER‐mitochondrial calcium homeostasis in the regulation of cellular proteostasis. Mutations that increase ER to mitochondrial calcium signaling lead to increased oxidative stress which promotes proteostatic collapse.
... Studies of LIN-12/Notch in C. elegans vulval development anticipated these key properties of T-ALL. Indeed, NRR-missense activating mutations in the ectodomain later associated with T-ALL were first observed in C. elegans LIN-12/Notch (Greenwald and Seydoux 1990), as was the dependence of signal transduction by these NRR-missense activated forms on the activities of -secretase (Levitan and Greenwald 1995) and ADAM protease (Wen et al. 1997). In addition, negative regulation by the conserved E3 ubiquitin ligase SEL-10/Fbw7 was also first observed via genetic interactions with mutant forms of LIN-12/Notch (Hubbard et al. 1997;Sundaram and Greenwald 1993). ...
... activity. This level of activity is enough to prevent the specification of the AC, so the inductive signal is not produced; however, there is insufficient constitutive activity to cause ligandindependent specification of the VPC 2 o fate (Greenwald et al., 1983;Levitan and Greenwald, 1995). The VPCs therefore behave like wild-type VPCs that have not received inductive signal, and all adopt the 3 o fate (Sternberg 2005). ...
Genetic analysis of LIN-12/Notch signaling in C. elegans has provided many insights into human biology. Activating missense mutations in the Negative Regulatory Region (NRR) of the ectodomain of LIN-12/Notch were first described in C. elegans, and similar mutations in human Notch were later found to cause T-cell acute lymphoblastic leukemia (T-ALL). The ubiquitin ligase sel-10/Fbw7 is the prototype of a conserved negative regulator of lin-12/Notch that was first defined by loss-of-function mutations that enhance lin-12 NRR-missense activity in C. elegans, and then demonstrated to regulate Notch activity in mammalian cells and to be a bona fide tumor suppressor in T-ALL. Here, we report the results of an RNAi screen of 248 C. elegans protein kinase-encoding genes with human orthologs for enhancement of a weakly activating NRR-missense mutation of lin-12 in the Vulval Precursor Cells. We identified, and validated, thirteen kinase genes whose loss led to increase lin-12 activity; eleven of these genes have never been implicated previously in regulating Notch activity in any system. Depleting the activity of five kinase genes (cdk-8, wnk-1, kin-3, hpo-11, and mig-15) also significantly enhanced the activity of a transgene in which heterologous sequences drive expression of the untethered intracellular domain of LIN-12, suggesting that they increase the activity or stability of the signal-transducing form of LIN-12/Notch. Precedents set by other regulators of lin-12/Notch defined through genetic interactions in C. elegans suggest that this new set of genes may include negative regulators that are functionally relevant to mammalian development and cancer.
... The C. elegans presenilin family encompasses three genes; hop-1, sel-12 and spe-4 [83][84][85]. Although hop-1 and sel-12 are widely expressed, including in muscle and neurons [86], the more distantly related, spe-4 is exclusively expressed in the male germ line [83]. ...
... sel-12 shows higher sequence identity to human presenilin compared to hop-1 and has been shown to localize to the endoplasmic reticulum [86]. sel-12 mutations were initially identified as suppressors that could alleviate developmental defects associated with excessive Notch signaling [84]. ...
Neurodegenerative diseases like Alzheimer’s disease (AD) are poised to become a global health crisis, and therefore understanding the mechanisms underlying the pathogenesis is critical for the development of therapeutic strategies. Mutations in genes encoding presenilin (PSEN) occur in most familial Alzheimer’s disease but the role of PSEN in AD is not fully understood. In this review, the potential modes of pathogenesis of AD are discussed, focusing on calcium homeostasis and mitochondrial function. Moreover, research using Caenorhabditis elegans to explore the effects of calcium dysregulation due to presenilin mutations on mitochondrial function, oxidative stress and neurodegeneration is explored.
... The C. elegans presenilin family encompasses three genes hop-1, sel-12 and spe-4 (Arduengo et al., 1998;Levitan and Greenwald, 1995;Li and Greenwald, 1997). Although hop-1 and sel-12 are widely expressed, including in muscle and neurons (Levitan and Greenwald, 1998), the more distantly related, spe-4 is exclusively expressed in the male germline (Arduengo et al., 1998). ...
... sel-12 shows higher sequence identity to human presenilin compared to hop-1 and has been shown to localize to the endoplasmic reticulum (Levitan and Greenwald, 1998). sel-12 mutations were initially identified as suppressors that could alleviate developmental defects associated with excessive Notch signaling (Levitan and Greenwald, 1995). ...
Neurodegenerative diseases like Alzheimer’s disease (AD) are poised to become a global health crisis, and therefore understanding the mechanisms underlying the pathogenesis is critical for the development of therapeutic strategies. Mutations in genes encoding presenilin occur in most familial Alzheimer’s disease but the role of PSEN in AD is not fully understood. In this review, the potential modes of pathogenesis of AD are discussed, focusing on calcium homeostasis and mitochondrial function. Moreover, research using Caenorhabditis elegans to explore the effects of calcium dysregulation due to presenilin mutations on mitochondrial function, oxidative stress and neurodegeneration is explored.
... TRN lysosomes, visualized as small puncta in day 1 adult animals, were distinguished by the colocalization of GFP and mCherry markers in neuronal cell bodies (Fig. 1A). We introduced these markers into three sel-12 mutants: sel-12(ty11), which contains a premature stop codon in sel-12 that leads to a predicted truncated protein and null mutant [35]; sel-12(ar131), which contains a missense mutation that is conserved with an fAD-linked presenilin mutation [36]; and sel-12(ok2078), a deletion mutant [17]. We found a significant increase in lysosome volume in the sel-12 mutant TRNs (Fig. 1A, B, S1A). ...
Compromised lysosome function is implicated in the pathology of many neurodegenerative diseases, including Alzheimer's disease (AD). Familial Alzheimer's disease (fAD) is caused primarily by mutations in the presenilin encoding genes, but the underlying mechanism remains obscure. Loss of the conserved C. elegans presenilin orthologue SEL-12 results in increased mitochondrial calcium, which promotes neurodegeneration. Here, we find that sel-12 mutant lysosomes, independent of SEL-12 proteolytic activity, are significantly enlarged and more alkaline due to increased ER-to-mitochondrial calcium signaling and concomitant mitochondrial oxidative stress. These defects and their dependence on mitochondrial calcium are recapitulated in human fAD fibroblasts, demonstrating a conserved role for mitochondrial calcium in presenilin-mediated lysosome dysfunction. sel-12 mutants also have increased contact surface area between the ER, mitochondria, and lysosomes, suggesting sel-12 has an additional role in modulating organelle contact and communication. Overall, we demonstrate that SEL-12 maintains lysosome acidity and lysosome health by controlling ER-to-mitochondrial calcium signaling.
... Although mutations in PSEN1 and PSEN2 lead to early-onset familial AD, their functional consequences are not yet well-defined [58]. In congruence with the mammalian studies, mutation of the PSEN ortholog sel-12 in C. elegans results in the dysregulation of ER Ca 2+ homeostasis [58,59]. Particularly, sel-12 mutants have elevated ER-mitochondrial Ca 2+ signaling, triggering increased mitochondrial Ca 2+ content, which subsequently leads to enhanced mitochondrial superoxide production. ...
In recent years, advances in science and technology have improved our quality of life, enabling us to tackle diseases and increase human life expectancy. However, longevity is accompanied by an accretion in the frequency of age-related neurodegenerative diseases, creating a growing burden, with pervasive social impact for human societies. The cost of managing such chronic disorders and the lack of effective treatments highlight the need to decipher their molecular and genetic underpinnings, in order to discover new therapeutic targets. In this effort, the nematode Caenorhabditis elegans serves as a powerful tool to recapitulate several disease-related phenotypes and provides a highly malleable genetic model that allows the implementation of multidisciplinary approaches, in addition to large-scale genetic and pharmacological screens. Its anatomical transparency allows the use of co-expressed fluorescent proteins to track the progress of neurodegeneration. Moreover, the functional conservation of neuronal processes, along with the high homology between nematode and human genomes, render C. elegans extremely suitable for the study of human neurodegenerative disorders. This review describes nematode models used to study neurodegeneration and underscores their contribution in the effort to dissect the molecular basis of human diseases and identify novel gene targets with therapeutic potential.
... [54] A C. elegans includes the following systems: an alimentary system, a reproductive system, a nervous system, and an excretory system, [55] and the detailed structures of the body are presented in Figure 7a. As a frequently used model animal, C. elegans has merits like transparency, rapid life cycle (3 d), short lifespan (2-3 weeks), constant cell number, invariant developmental trajectory, and well-annotated genome, [56,57] and hence it has been widely used in studies of Alzheimer's disease, [58,59] human metabolic regulation, [60] nanotoxicology, [61] bioimaging, [62,63] and genomics. [64,65] In this work, we chose C. elegans as a representative model for evaluating the in vivo imaging performance of CPCDs. ...
Nucleolus, which participates in many crucial cellular activities, is an ideal target for evaluating the state of a cell or an organism. Here, bright red‐emissive carbon dots (termed CPCDs) with excitation‐independent/polarity‐dependent fluorescence emission are synthesized by a one‐step hydrothermal reaction between congo red and p‐phenylenediamine. The CPCDs can achieve wash‐free, real‐time, long‐term, and high‐quality nucleolus imaging in live cells, as well as in vivo imaging of two common model animals—zebrafish and Caenorhabditis elegans (C. elegans). Strikingly, CPCDs realize the nucleolus imaging of organs/flowing blood cells in zebrafish at a cellular level for the first time, and the superb nucleolus imaging of C. elegans suggests that the germ cells in the spermatheca probably have no intact nuclei. These previously unachieved imaging results of the cells/tissues/organs may guide the zebrafish‐related studies and benefit the research of C. elegans development. More importantly, a novel strategy based on CPCDs for in vivo toxicity evaluation of materials/drugs (e.g., Ag⁺), which can visualize the otherwise unseen injuries in zebrafish, is developed. In conclusion, the CPCDs represent a robust tool for visualizing the structures and dynamic behaviors of live zebrafish and C. elegans, and may find important applications in cell biology and toxicology.
... Caenorhabditis elegans can distinguish between different fragrances, foods, and temperatures, and respond accordingly under the control of various neurotransmitters (Calhoun et al., 2015). The first C. elegans AD model, based on the amyloid cascade hypothesis, was generated in 1995 (Levitan & Greenwald, 1995). In this model, the Aβ peptide is tagged with a secretion signaling sequence through the expression of the unc-54 promoter in the body wall muscles, resulting in a paralysis phenotype for quantitative analysis. ...
Although great advances in elucidating the molecular basis and pathogenesis of Alzheimer's disease (AD) have been made and multifarious novel therapeutic approaches have been developed, AD remains an incurable disease. Evidence shows that AD neuropathology occurs decades before clinical presentation. AD is divided into three stages: preclinical stage, mild cognitive impairment (MCI), and AD dementia. In the natural world, some animals, such as non-human primates (NHPs) and canines, can develop spontaneous AD-like dementia. However, most animals do not develop AD. With the development of transgenic techniques, both invertebrate and vertebrate animals have been employed to uncover the mechanisms of AD and study treatment methods. Most AD research focuses on early-onset familial AD (FAD) because FAD is associated with specific genetic mutations. However, there are no well-established late-onset sporadic AD (SAD) animal models because SAD is not directly linked to any genetic mutation, and multiple environmental factors are involved. Moreover, the widely used animal models are not able to sufficiently recapitulate the pathological events that occur in the MCI or preclinical stages. This review summarizes the common models used to study AD, from yeast to NHP models, and discusses the different applications, evaluation methods, and challenges related to AD animal models, as well as prospects for the evolution of future studies.
... Increased protein carbonyl content [154], neuronal lipid peroxidation [155] and increased ROS levels [99] have been reported in transgenic worms expressing Aβ. Furthermore, mutations in the sel-12 gene that encodes for presenilins in C. elegans [156], which are related to the excessive production of Aβ1-42 in early-onset AD, can lead to cell death via oxidative stress [157]. In addition, pathogenic protein aggregation in cells has been associated with an excessive production of ROS because of their effects in proteins, such as amino-acid side chain oxidation, hydroperoxide formation, the carbonylation of proteins and the fragmentation of the protein backbone [158]. ...
Alzheimer’s disease (AD) is an age-dependent, progressive disorder affecting millions of people. Currently, the therapeutics for AD only treat the symptoms. Although they have been used to discover new products of interest for this disease, mammalian models used to investigate the molecular determinants of this disease are often prohibitively expensive, time-consuming and very complex. On the other hand, cell cultures lack the organism complexity involved in AD. Given the highly conserved neurological pathways between mammals and invertebrates, Caenorhabditis elegans has emerged as a powerful tool for the investigation of the pathophysiology of human AD. Numerous models of both Tau- and Aβ-induced toxicity, the two prime components observed to correlate with AD pathology and the ease of performing RNA interference for any gene in the C. elegans genome, allow for the identification of multiple therapeutic targets. The effects of many natural products in main AD hallmarks using these models suggest promising health-promoting effects. However, the way in which they exert such effects is not entirely clear. One of the reasons is that various possible therapeutic targets have not been evaluated in many studies. The present review aims to explore shared therapeutical targets and the potential of each of them for AD treatment or prevention.
... Notably, APP processing is an evolutionarily conserved biological pathway across different species (Shariati & De Strooper, 2013). Caenorhabditis elegans: In C. elegans, homologues of α-secretase orthologs (SUP-17, ADM-4), presenilin 1 (sel12, hop1), and nicastrin (aph-2) have been identified; however, a β-secretase homologue has not been identified, suggesting that APL-1 undergoes the α/γsecretase-mediated processing pathway (Goutte, Hepler, Mickey, & Priess, 2000;Jarriault & Greenwald, 2005;Levitan & Greenwald, 1995;Li & Greenwald, 1997;Link, 2006;McColl et al., 2012;Yu et al., 2000). Genetic screening in C. elegans has led to the discovery of the interaction of pen-2 and aph-1 with sel12 and aph-2; further, these are essential for γ-secretase activity (Francis et al., 2002;Goutte, Tsunozaki, Hale, & Priess, 2002). ...
Amyloid precursor protein (APP) is an evolutionarily conserved transmembrane protein and a well-characterized precursor protein of amyloid-beta (Aβ) peptides, which accumulate in the brains of individuals with Alzheimer’s disease (AD)-related pathologies. Aβ has been extensively investigated since the amyloid hypothesis in AD was proposed. Besides Aβ, previous studies on APP and its proteolytic cleavage products have suggested their diverse pathological and physiological functions. However, their roles still have not been thoroughly understood. In this review, we extensively discuss the evolutionarily-conserved biology of APP, including its structure and processing pathway, as well as recent findings on the physiological roles of APP and its fragments in the central nervous system and peripheral nervous system. We have also elaborated upon the current status of APP-targeted therapeutic approaches for AD treatment by discussing inhibitors of several proteases participating in APP processing, including α-, β-, and γ-secretases. Finally, we have highlighted the future perspectives pertaining to further research and the potential clinical role of APP.
... 41 Loss of apl-1, as well as its overexpression, produces adverse developmental effects, including larval lethality, which may be rescued by the neuronal expression of this protein's extracellular domain. 41 A reduction in the activity of sel-12, a presenilin homolog discovered in 1995 as a suppressor of a lin-12 gain-of-function mutation, 42 has also been reported to partially rescue apl-1 lethality, generally mimicking the regulatory relationship between human PSEN and APP. 41 More recently, the effects of agealtered metal homeostasis on the deposition of Aβ, as well as other late-onset symptoms like paralysis, have been studied in C. elegans models of AD. 43 Directed expression of the human Aβ peptide in Drosophila, despite the presence of the APP-homolog Appl, has been used to study Aβ toxicity in vivo, where it causes locomotive and cognitive defects in the flies. ...
Alzheimer's disease and Parkinson's disease are two of the most prevalent and disabling neurodegenerative diseases globally. Both are proteinopathic conditions and while occasionally inherited, are largely sporadic in nature. Although the advances in our understanding of the two have been significant, they are far from complete and neither diagnosis nor the current practices in treatment and rehabilitation is adequately helpful. Animal models have historically found application as testing beds for novel therapeutics and continue to be valuable aids in pharmacological research. This review chronicles the development of those models in the context of Alzheimer's and Parkinson's disease, and highlights the shifting paradigms in studying two human‐specific conditions in non‐human organisms. Cellular and molecular changes in neuronal cells may induce proteinopathies, leading to Alzheimer's and/or Parkinson's diseases (AD and PD respectively). Both AD and PD are marked by the loss of neurons due to cell death and result in the loss of cognition and impaired movement of patients. This review discusses the etiology of AD and PD in terms of historical perspectives known for these diseases.
... The advent of molecular genetics enabled fine dissection of the Notch signaling pathway and its importance in development and disease [41][42][43][44][45][46] . For example, seminal discoveries revealing the function and processing of the GLP-1 and LIN-12 Notch receptors in C. elegans have contributed considerably to our understanding of this important signaling pathway 8,27,[46][47][48][49][50] . In the context of the C. elegans germline, it has been known for decades that GLP-1 signaling intensity is important for controlling mitotic cell fate 8,14 . ...
Communication between the soma and germline optimizes germ cell fate programs. Notch receptors are key determinants of germ cell fate but how somatic signals direct Notch-dependent germ cell behavior is undefined. Here we demonstrate that SDN-1 (syndecan-1), a somatic transmembrane proteoglycan, controls expression of the GLP-1 (germline proliferation-1) Notch receptor in the Caenorhabditis elegans germline. We find that SDN-1 control of a somatic TRP calcium channel governs calcium-dependent binding of an AP-2 transcription factor (APTF-2) to the glp-1 promoter. Hence, SDN-1 signaling promotes GLP-1 expression and mitotic germ cell fate. Together, these data reveal SDN-1 as a putative communication nexus between the germline and its somatic environment to control germ cell fate decisions.
... The presenilins play a role in proteolytic cleavage within the transmembrane domains of other proteins such as Notch (11)(12)(13)(14), -amyloid precursor protein (APP) (15)(16)(17)(18)(19)(20), and endoplasmic reticulum stress sensor/endoplasmic reticulum resident transmembrane kinase (Ire1p) (21,22). The precise role of PS1 in this unusual form of proteolytic processing is unknown. ...
Presenilin (PS1 and PS2) holoproteins are transiently incorporated into low molecular weight (MW) complexes. During subsequent incorporation into a higher MW complex, they undergo endoproteolysis to generate stable N- and C-terminal fragments. Mutation of either of two conserved aspartate residues in transmembrane domains inhibits both presenilin-endoproteolysis and the proteolytic processing of β-amyloid precursor protein and Notch. We show that although PS1/PS2 endoproteolysis is not required for inclusion into the higher MW N- and C-terminal fragment-containing complex, aspartate mutant holoprotein presenilins are not incorporated into the high MW complexes. Aspartate mutant presenilin holoproteins also preclude entry of endogenous wild type PS1/PS2 into the high MW complexes but do not affect the incorporation of wild type holoproteins into lower MW holoprotein complexes. These data suggest that the loss of function effects of the aspartate mutants result in altered PS complex maturation and argue that the functional presenilin moieties are contained in the high molecular weight complexes.
... Aβ accumulation is an outcome of sequential enzymatic processing of the human amyloid precursor protein (APP) by membrane-bound secretase enzymes mediated by the subcomponent PS1 and PS2 presenilin proteins. Although the product of the worm sel-12 gene provided some of the first evidence for presenilins as functional subunits of human γ-secretase (Levitan and Greenwald, 1995), the C. elegans genome lacks both a clear β-secretase ortholog and an ortholog of APP that would be processed to yield Aβ peptides. The worm APL-1 protein is an ortholog of the human amyloid beta precursor-like proteins 1 and 2 (APLP1 and APLP2), and has 71% sequence similarity to the intracellular domain of APP, but completely lacks an Aβ domain. ...
The global burden of neurodegenerative diseases underscores the urgent need for innovative strategies to define new drug targets and disease-modifying factors. The nematode Caenorhabditis elegans has served as the experimental subject for multiple transformative discoveries that have redefined our understanding of biology for ∼60 years. More recently, the considerable attributes of C. elegans have been applied to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease and Huntington's disease. Transgenic nematodes with genes encoding normal and disease variants of proteins at the single- or multi-copy level under neuronal-specific promoters limits expression to select neuronal subtypes. The anatomical transparency of C. elegans affords the use of co-expressed fluorescent proteins to follow the progression of neurodegeneration as the animals age. Significantly, a completely defined connectome facilitates detailed understanding of the impact of neurodegeneration on organismal health and offers a unique capacity to accurately link cell death with behavioral dysfunction or phenotypic variation in vivo . Moreover, chemical treatments, as well as forward and reverse genetic screening, hasten the identification of modifiers that alter neurodegeneration. When combined, these chemical-genetic analyses establish critical threshold states to enhance or reduce cellular stress for dissecting associated pathways. Furthermore, C. elegans can rapidly reveal whether lifespan or healthspan factor into neurodegenerative processes. Here, we outline the methodologies employed to investigate neurodegeneration in C. elegans and highlight numerous studies that exemplify its utility as a pre-clinical intermediary to expedite and inform mammalian translational research.
... Studies of presenilin are well-founded in C. elegans research: the first cellular functions of presenilin were determined by their homology to the C. elegans protein, SEL-12. Levitan and Greenwald (1995) showed that sel-12 was involved in the Notch pathway to mediate cell fate decisions during development. Over-expression of both human PSEN1 and PSEN2 rescued a Notch-dependent egg-laying phenotype shown by sel-12(ar171)suggesting a conserved function between human presenilin and C. elegans sel-12 (Levitan et al., 1996). ...
Since Caenorhabditis elegans was first introduced as a genetic model organism by Sydney Brenner, researchers studying it have made significant contributions in numerous fields including investigations of the pathophysiology of neurodegenerative diseases. The simple anatomy, optical transparency, and short life-span of this small nematode together with the development and curation of many openly shared resources (including the entire genome, cell lineage and the neural map of the animal) allow researchers using C. elegans to move their research forward rapidly in an immensely collaborative community. These resources have allowed researchers to use C. elegans to study the cellular processes that may underlie human diseases. Indeed, many disease-associated genes have orthologs in C. elegans, allowing the effects of mutations in these genes to be studied in relevant and reproducible neuronal cell-types at single-cell resolution in vivo. Here we review studies that have attempted to establish genetic models of specific human neurodegenerative diseases (ALS, Alzheimer’s Disease, Parkinson’s Disease, Huntington’s Disease) in C. elegans and what they have contributed to understanding the molecular and genetic underpinnings of each disease. With continuous advances in genome engineering, research conducted using this small organism first established by Brenner, Sulston and their contemporaries will continue to contribute to the understanding of human nervous diseases.
... The advent of molecular genetics enabled fine dissection of the Notch signaling pathway and its importance in development and disease (Andersson et al., 2011;Greenwald, 1985;Greenwald et al., 1983;Kidd et al., 1986;Kopan and Ilagan, 2009;Wharton et al., 1985). For example, seminal discoveries revealing the function and processing of the GLP-1 and LIN-12 Notch receptors in C. elegans have contributed considerably to our understanding of this important signaling pathway Kimble, 1987, 1989;Evans et al., 1994;Greenwald et al., 1983;Levitan and Greenwald, 1995;Shaye and Greenwald, 2002;Yochem and Greenwald, 1989). ...
Notch receptors are essential membrane-bound regulators of cell proliferation and differentiation in metazoa. In the nematode Caenorhabditis elegans, correct expression of GLP-1 (germline proliferation-1), a germline-expressed Notch receptor, is important for germ cell maintenance. However, mechanisms that regulate GLP-1 expression are undefined. Here, we demonstrate that an AP-2 transcription factor (APTF-2) regulates GLP-1 expression through calcium-dependent binding to a conserved motif in the glp-1 promoter. Our data reveals that SDN-1 (syndecan-1), a transmembrane proteoglycan, regulates a TRP calcium channel in the soma to modulate the interaction between APTF-2 and glp-1 promoter - thus providing a potential communication nexus between the germline and its somatic environment to control germ cell fate decisions.
... Incorrect cleavage of APP results in Ap-42 production and the subsequent formation of the amyloid plaques that characterize the human disorder. Study of this pathway in C elegans, using enhancer/suppressor screens, has helped identify interacting genes and provide insight into the mechanism of disease pathogenesis (Levitan and Greenwald 1995;Okochi et al. 2000;Driscoll and Gerstbrein 2003). ...
The neuronal ceroid lipofuscinoses (Batten disease) are a group of inherited neurological disorders which predominantly affect children. They are characterized by the accumulation of autofluorescent storage material in lysosomes and occur with a frequency between 1 in 12 500 and 1 in 100 000 births. To date, six genes underlying different sub-types of the disease have been cloned and many studies in cell culture and mouse models performed. However the mechanism of disease pathogenesis remains poorly understood. The simple nematode worm, Caenorhabditis elegans, has a fully sequenced genome, completely mapped cell lineage and nervous system, is easy to maintain and manipulate, and is an organism about which much information, including the results of many genome wide studies, is available. It is thus a good model organism for the study of genes that underlie human neurological disorders. The aim of this work was to investigate whether C. elegans could be used as a model system for investigating the function of NCL genes and the pathological mechanisms that underlie disease manifestation. Homologues to PPT1 and CLN3 were identified in C. elegans and their expression confirmed. Mutation of PPT1 underlies the most severe NCL type, infantile NCL (INCL). Further analysis of CePPT-1 determined a high level of sequence and structural similarity to the human enzyme and demonstrated that it could perform the same catalytic reaction under the same conditions. Analysis of a ppt-1 null mutant (MN1) identified a phenotype of developmental delay, defective egg laying and grossly abnormal mitochondrial morphology. A homologue to the enzyme, acyl-protein thioesterase-1 was also identified (ATH-1) and expression confirmed. The role of PPT-1 in the cell and how this may relate to the pathogenesis of INCL and other NCL types is presented.
... La PSé1 est exprimée dans le SNC ainsi que dans les tissus périphériques. Il s'agit d'une protéine très conservée au cours de l'évolution dont on a trouvé des gènes orthologues aussi bien chez le nématode Cenorhabditis elegans(Levitan and Greenwald, 1995) que chez la mouche du vinaigre Drosophila melanogaster(van de Hoef et al., 2009).Dans la cellule, la PSé1 est principalement localisée dans les membranes intracellulaires de l'enveloppe périnucléaire, dans les membranes du RE et dans celles de l'appareil de ...
Les maladies neurodégénératives touchent plus de 33 millions de personnes dans le monde. Elles sont caractérisées par la dégénérescence progressive des neurones et un dysfonctionnement dans le cerveau et/ou la moëlle épinière. Leurs mécanismes (perte des cellules neuronales, axonopathie, perte synaptique, inflammation, dysfonction immunitaire...) ont une incidence sur les fonctions moteurs et cognitives et peuvent générer une dépendance croissante des malades. Le traitement de la plupart des maladies neurodégénératives reste à l'heure actuelle essentiellement symptomatique ; s'il permet d'améliorer la qualité de vie des patients, il est le plus souvent sans effet sur l'évolution de la maladie. L’ensemble de nos résultats a permis de mettre en évidence des mécanismes physiopathologiques inédits de la maladie d’Alzheimer et la sclérose latérale amyotrophique, et d’imaginer de nouvelles pistes thérapeutiques. Nous avons étudié les effets toxiques des AβO, sur un modèle in vitro de neurones primaires ainsi que sur un modèle de coculture nerf muscle reproduisant des jonctions neuromusculaires, en nous concentrant sur leur cinétique de développement, puis sur leur mécanismes moléculaires et cellulaires. Nous avons pu avancer l’hypothèse d’un lien de causalité étroit entre le glutamate et les AβO aussi bien dans la maladie d’Alzheimer que dans la sclérose latérale amyotrophique. Il a été mis en évidence que des modèles de culture cellulaire permettaient de répondre à des questions ciblées, dans la mesure où ils reproduisent certains phénomènes neuropathologiques observés chez les malades.
... Beta-amyloid peptide precursor protein and the presenilins, PS1 and PS2 were the first ADassociated proteins identified. C. elegans presenilin orthologs sel-12, hop-1 and spe-4 (Baumeister et al., 1997;Levitan and Greenwald, 1995;Li and Greenwald, 1997;Smialowska and Baumeister, 2006) linked AD to the apoptotic pathway (Kitagawa et al., 2003) and Notch signaling (Berezovska et al., 1998;. Mutations in worm orthologs of the presenilin proteases responsible for cleaving APP through the γ-secretase pathway, favored in the pathogenesis of AD (Nunan and Small, 2000;Selkoe, 1999), such as a sel-12 mutation, have revealed deficits in thermal memory necessary for proper thermotaxis, a behavior that is dependent upon the AFD sensory neurons and AIZ and AIY interneurons. ...
... A few years after their discovery, it was shown that PSENs encode proteins that, acting as aspartyl proteases, sustain γ-secretase cleavage of APP to produce Aβ peptides [50,51]. PSEN genes are highly conserved during evolution having homologs in organisms as distant as Caenorhabditis elegans [52], Drosophila melanogaster [53], and lower chordates [54]. ...
Alzheimer’s disease (AD) is the most common form of dementia. Even though most AD cases are sporadic, a small percentage is familial due to autosomal dominant mutations in amyloid precursor protein (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) genes. AD mutations contribute to the generation of toxic amyloid β (Aβ) peptides and the formation of cerebral plaques, leading to the formulation of the amyloid cascade hypothesis for AD pathogenesis. Many drugs have been developed to inhibit this pathway but all these approaches currently failed, raising the need to find additional pathogenic mechanisms. Alterations in cellular calcium (Ca2+) signaling have also been reported as causative of neurodegeneration. Interestingly, Aβ peptides, mutated presenilin-1 (PS1), and presenilin-2 (PS2) variously lead to modifications in Ca2+ homeostasis. In this contribution, we focus on PS2, summarizing how AD-linked PS2 mutants alter multiple Ca2+ pathways and the functional consequences of this Ca2+ dysregulation in AD pathogenesis.
... Presenilins were also quickly connected to signaling from the Notch family of cell-surface receptors. Through genetic screening, the C. elegans gene sel-12, related to spe-4 and human PSEN1 and PSEN2, was found to facilitate signaling from lin-12 and glp-1, worm Notch receptor orthologs [37]. Moreover, knockout of PSEN1 in mice resulted in an embryonic lethal phenotype closely resembling that seen upon knockout of Notch1 [15,16]. ...
γ-Secretase was initially defined as a proteolytic activity that cleaves within the transmembrane of the amyloid precursor protein (APP) to produce the amyloid β-peptide of Alzheimer's disease. The discovery of mutations in APP and the presenilins associated with familial Alzheimer's disease and their effects on APP processing dovetailed with pharmacological studies on γ-secretase, leading to the revelation that presenilins are unprecedented membrane-embedded aspartyl proteases. Other members of what became known as the γ-secretase complex were subsequently identified. In parallel with these advances, connections between presenilins and Notch receptors essential to metazoan development became evident, resulting in the concurrent realization that γ-secretase also carries out intramembrane proteolysis of Notch as part of its signaling mechanism. Substantial progress has been made toward elucidating how γ-secretase carries out complex processing of transmembrane domains, how it goes awry in familial Alzheimer's disease, the scope of its substrates, and the atomic details of its structure. Critical questions remain for future study, toward further unraveling the complexity of this unique membrane-embedded proteolytic machine and its roles in biology and disease.
... The region between the LNR domain and the transmembrane domain is also highly conserved across evolution and is referred to as the HD Notch is often referred to as NEXT (Notch extracellular truncation) and contains a very short extracellular domain, the TMD and NICD. NEXT is subjected to subsequent S3 (site-3) cleavage by the γ-secretase complex (De Strooper et al., 1999;Levitan & Greenwald, 1995;Struhl & Greenwald, 1999;Ye, Lukinova, & Fortini, 1999). Ca 2+ ...
Notch signaling is involved in the development of almost all organ systems and is required post-developmentally to modulate tissue homeostasis. Rare variants in Notch signaling pathway genes are found in patients with rare Mendelian disorders, while unique or recurrent somatic mutations in a similar set of genes are identified in cancer. The human genome contains four genes that encode Notch receptors, NOTCH1-4, all of which are linked to genetic diseases and cancer. Although some mutations have been classified as clear loss- or gain-of-function alleles based on cellular or rodent based assay systems, the functional consequence of many variants/mutations in human Notch receptors remain unknown. In this review, I will first provide an overview of the domain structure of Notch receptors and discuss how each module is known to regulate Notch signaling activity in vivo using the Drosophila Notch receptor as an example. Next, I will introduce some interesting mutant alleles that have been isolated in the fly Notch gene over the past > 100 years of research and discuss how studies of these mutations have facilitated the understanding of Notch biology. By identifying unique alleles of the fly Notch gene through forward genetic screens, mapping their molecular lesions and characterizing their phenotypes in depth, one can begin to unravel new mechanistic insights into how different domains of Notch fine-tune signaling output. Such information can be useful in deciphering the functional consequences of rare variants/mutations in human Notch receptors, which in turn can influence disease management and therapy.
... Following ligand-dependent site 2 cleavage, g-secretase performs cleavage at site 3 in the transmembrane domain. C. elegans g-secretase is composed of catalytic subunit paralogs SEL-12 and HOP-1, which are orthologous to mammalian Presenilin 1 and 2, and associated subunits APH-1, APH-2, and PEN-2, which are orthologous to mammalian Aph1, Nicastrin, and Pen2, respectively (Levitan and Greenwald 1995;Li and Greenwald 1997;Westlund et al. 1999;Goutte et al. 2000Goutte et al. , 2002Levitan et al. 2001;Francis et al. 2002). SEL-12 and HOP-1 act redundantly in GLP-1 signaling during larval development, with HOP-1 functioning predominantly in adulthood (Agarwal et al. 2018). ...
Stem cell systems regulate tissue development and maintenance. The germline stem cell system is essential for animal reproduction, controlling both the timing and number of progeny through its influence on gamete production. In this review, we first draw general comparisons to stem cell systems in other organisms, and then present our current understanding of the germline stem cell system in Caenorhabditis elegans In contrast to stereotypic somatic development and cell number stasis of adult somatic cells in C. elegans, the germline stem cell system has a variable division pattern, and the system differs between larval development, early adult peak reproduction and age-related decline. We discuss the cell and developmental biology of the stem cell system and the Notch regulated genetic network that controls the key decision between the stem cell fate and meiotic development, as it occurs under optimal laboratory conditions in adult and larval stages. We then discuss alterations of the stem cell system in response to environmental perturbations and aging. A recurring distinction is between processes that control stem cell fate and those that control cell cycle regulation. C. elegans is a powerful model for understanding germline stem cells and stem cell biology.
... The genetic model organism Caenorhabditis elegans is a powerful system for the functional analysis of disease-associated genetic variation, particularly for high-throughput in vivo characterization of risk genes identified through genomics (27). C. elegans's fully sequenced and thoroughly annotated genome and complete connectome have fueled numerous disease discoveries, including the identification of presenilins as part of the gamma secretase complex and the role of the insulin signaling pathway in normal and pathological aging (27)(28)(29)(30). There are clear C. elegans orthologs for >50% of human genes, and human genes have repeatedly been shown to be so structurally and functionally conserved that they can directly replace their C. elegans ortholog (27,(31)(32)(33). ...
A major challenge facing the genetics of autism spectrum disorders (ASDs) is the large and growing number of candidate risk genes and gene variants of unknown functional significance. Here, we used Caenorhabditis elegans to systematically functionally characterize ASD-associated genes in vivo. Using our custom machine vision system, we quantified 26 phenotypes spanning morphology, locomotion, tactile sensitivity, and habituation learning in 135 strains each carrying a mutation in an ortholog of an ASD-associated gene. We identified hundreds of genotype–phenotype relationships ranging from severe developmental delays and uncoordinated movement to subtle deficits in sensory and learning behaviors. We clustered genes by similarity in phenomic profiles and used epistasis analysis to discover parallel networks centered on CHD8•chd-7 and NLGN3•nlg-1 that underlie mechanosensory hyperresponsivity and impaired habituation learning. We then leveraged our data for in vivo functional assays to gauge missense variant effect. Expression of wild-type NLG-1 in nlg-1 mutant C. elegans rescued their sensory and learning impairments. Testing the rescuing ability of conserved ASD-associated neuroligin variants revealed varied partial loss of function despite proper subcellular localization. Finally, we used CRISPR-Cas9 auxin-inducible degradation to determine that phenotypic abnormalities caused by developmental loss of NLG-1 can be reversed by adult expression. This work charts the phenotypic landscape of ASD-associated genes, offers in vivo variant functional assays, and potential therapeutic targets for ASD.
... Concurrently, genetic studies in C. elegans and mice connected presenilins to the Notch family of cell-surface receptors. [25][26][27] Signaling from Notch is essential for the determination of cell fate in embryogenesis in all metazoans, 28 and knockout of presenilins leads to embryonic-lethal phenotypes similar to that seen upon knockout of Notch1. 26,27 Signaling from Notch1 was found to be dependent on the release of its intracellular domain through cleavage within the TMD of the receptor. ...
γ-Secretase is a membrane-embedded protease complex, with presenilin as the catalytic component containing two transmembrane aspartates in the active site. With over 90 known substrates, the γ-secretase complex is considered "the proteasome of the membrane", with central roles in biology and medicine. The protease carries out hydrolysis within the lipid bilayer to cleave the transmembrane domain of substrate multiple times before releasing secreted products. For many years, elucidation of γ-secretase structure and function largely relied on small molecule probes and mutagenesis. Recently however, advances in cryo-electron microscopy have led to the first detailed structures of the protease complex. Two new reports of structures of γ-secretase bound to membrane protein substrates provide great insight into the nature of substrate recognition and how Alzheimer-causing mutations in presenilin might alter substrate binding and processing. These new structures offer a powerful platform for elucidating enzyme mechanisms, deciphering effects of disease-causing mutations, and advancing Alzheimer drug discovery.
... Presenilin (PS)1 and 2 are multi-pass transmembrane (TM) proteins of aspartyl protease family. Their primary sequence is evolutionarily conserved in eukaryotes, having homologues in organisms as distant as C. elegans (Levitan and Greenwald, 1995), Drosophila (Boulianne et al., 1997), and lower chordates (Martínez-Mir et al., 2001), suggesting functional conservation. Mammalian PS1 and 2 share extensive amino acid sequence identity and are synthesized as 50 kDa polypeptides (Hutton and Hardy, 1997). ...
In our previous report, we showed that Presenilin (PS)1 and 2 have differential expression profile from early embryonic stages till adulthood in mouse cerebral cortex, suggesting that both of these proteins are crucial for brain development. Genetic manipulation studies have also shown the involvement of PS1 in brain development, but PS2 remains largely unexplored. In order to understand how PS1 and 2 mediate developmental functions, we have investigated the interaction of PS1 and 2 with proteins of mouse cere-bral cortex during development. Co-immunoprecipitation (Co-IP) combined with MALDI-MS/MS analysis revealed 12 interacting partners of PS1 and 11 partners of PS2. The interacting proteins were different for PS1 and 2, and involved in cell division, glycolysis, cell adhesion and protein trafficking. Densitometric analysis of protein bands visualized after SDS-PAGE separation of Co-IP proteins revealed variation in their amount and degree of interaction during different developmental stages of mice. Further, immunoblot based validation of PS1 interacting protein Notch-1 showed maximum interaction at embryonic day (E) 12.5, decline at E18.5, upregulation from postnatal day 0 (P0) to P20 and thereafter reduction at P45 and 20 weeks. In-silico analysis of PS and its interacting proteins indicated conformation based interaction through common type of secondary structures having alpha helical, extended beta strand and random coil, and CK2, PKC phosphorylation and myristoylation motifs. Taken together, our study showed that PS1 and PS2 interact to varying extent with different proteins of mouse cerebral cortex and suggests their interaction based on specific conformation and involvement in diverse functions essential for the brain development.
... The PSEN-1 gene encodes a protein component of the gammasecretase complex involved in the processing of the APP thus PSEN plays a very direct role in the proteolytic processing of APP [26]. Functionally, PSN-1 is involved in a numerous fundamental mechanisms of molecular pathways [27][28][29], regulation of β-amyloid precursor protein processing [30], regulation of transport [31], regulation of intracellular calcium homeostasis [32], stabilization of the cytoskeleton [33], which when disrupted lead to the AD. Mutations of the PSN lead to chromosomal instability and trisomy 21 mosaicism in AD patients [34] and consequently account for up to 50% of all familial AD cases [35]. ...
Although age is a dominant risk factor for Alzheimer's disease (AD), epidemiological studies have shown that physical activity may significantly decrease age-related risks for AD, and indeed mitigate the impact in existing diagnosis. The aim of this study was to perform a narrative review on the preventative, and mitigating, effects of physical activity on AD onset, including genetic factors, mechanism of action and physical activity typology. In this article, we conducted a narrative review of the influence physical activity and exercise have on AD, utilising key terms related to AD, physical activity, mechanism and prevention, searching the online databases; Web of Science, PubMed and Google Scholar, and, subsequently, discuss possible mechanisms of this action. On the basis of this review, it is evident that physical activity and exercise may be incorporated in AD, notwithstanding, a greater number of high-quality randomised controlled trials are needed, moreover, physical activity typology must be acutely considered, primarily due to a dearth of research on the efficacy of physical activity types other than aerobic.
... Transgenic expression of human genes is routinely done to confirm functional conservation and to observe the effects of disease-associated mutations. Notable examples of the utility of C. elegans to determine conserved human gene functions relevant to disease include the identification of presenilins as part of the gamma secretase complex, the mechanism of action of selective serotonin reuptake inhibitors, and the role of the insulin signaling pathway in normal and pathological ageing ( Kaletta and Hengartner, 2006;Levitan and Greenwald, 1995;Levitan et al., 1996;Murphy et al., 2003;Ranganathan et al., 2001). ...
Our ability to sequence genomes has vastly surpassed our ability to interpret the genetic variation we discover. This presents a major challenge in the clinical setting, where the recent application of whole-exome and whole-genome sequencing has uncovered thousands of genetic variants of uncertain significance. Here, we present a strategy for targeted human gene replacement and phenomic characterization, based on CRISPR-Cas9 genome engineering in the genetic model organism Caenorhabditis elegans, that will facilitate assessment of the functional conservation of human genes and structure-function analysis of disease-associated variants with unprecedented precision. We validate our strategy by demonstrating that direct single-copy replacement of the C. elegans ortholog (daf-18) with the critical human disease-associated gene phosphatase and tensin homolog (PTEN) is sufficient to rescue multiple phenotypic abnormalities caused by complete deletion of daf-18, including complex chemosensory and mechanosensory impairments. In addition, we used our strategy to generate animals harboring a single copy of the known pathogenic lipid phosphatase inactive PTEN variant (PTEN-G129E), and showed that our automated in vivo phenotypic assays could accurately and efficiently classify this missense variant as loss of function. The integrated nature of the human transgenes allows for analysis of both homozygous and heterozygous variants and greatly facilitates high-throughput precision medicine drug screens. By combining genome engineering with rapid and automated phenotypic characterization, our strategy streamlines the identification of novel conserved gene functions in complex sensory and learning phenotypes that can be used as in vivo functional assays to decipher variants of uncertain significance.
... We normalized the fluorescence intensity of the GCaMP6 to that of mCherry to determine the relative mitochondrial matrix Ca 2+ concentration in day one adult wild type animals and two sel-12 mutants, sel-12 (ar131) and sel-12(ty11). sel-12(ar131) mutants carry a missense mutation in the sel-12 gene, which is a conserved change seen in FAD patients (ALZFORUM, 2016;Levitan and Greenwald, 1995) and sel-12 (ty11) mutants contain a premature stop codon in the sel-12 gene, which is a predicted null mutation (Cinar et al., 2001). Strikingly, we observe that the relative fluorescence intensity of GCaMP6 to mCherry is 2.4 and 3.4 fold higher in sel-12 (ar131) and sel-12(ty11) animals, respectively, compared to wild type animals ( Figure 1A). ...
Mitochondrial dysfunction and subsequent metabolic deregulation is observed in neurodegenerative diseases and aging. Mutations in the presenilin PSEN encoding genes PSEN1 and PSEN2 cause most cases of familial Alzheimer s disease AD; however, the underlying mechanism of pathogenesis remains unclear. Here, we show that mutations in the C. elegans gene encoding a PSEN homolog, sel-12 result in mitochondrial metabolic defects that promote neurodegeneration as a result of oxidative stress. In sel-12 mutants, elevated endoplasmic reticulum ER-mitochondrial Ca²⁺signaling leads to an increase in mitochondrial Ca²⁺ content which stimulates mitochondrial respiration resulting in an increase in mitochondrial superoxide production. By reducing ER Ca²⁺ release, mitochondrial Ca²⁺uptake or mitochondrial superoxides in sel-12 mutants, we demonstrate rescue of the mitochondrial metabolic defects and prevent neurodegeneration. These data suggest that mutations in PSEN alter mitochondrial metabolic function via ER to mitochondrial Ca2+ signaling and provide insight for alternative targets for treating neurodegenerative diseases.
... Subsequent cleavage of the remaining C-terminal fragment (CTF) by the γ-secretase releases the intracellular region into the cytoplasm, the released peptide is a functional unit and therefore termed intracellular domain (ICD), also referred to as the released ICD in the following text (Brown et al., 2000;Lal and Caplan, 2011;Saftig and Lichtenthaler, 2015). Notch was the first-reported, well-characterized membrane-protein undergoing RIP (Levitan and Greenwald, 1995;Blaumueller et al., 1997;De Strooper et al., 1999) and the released Notch ICD exhibits defined cellular functions, acting as a transcription factor (Bailey and Posakony, 1995;Baker and Zitron, 1995). Although release of the NG2 ectodomain had been previously documented (Nishiyama et al., 1995;Deepa et al., 2006), our published work demonstrated that NG2 undergoes RIP, generating a CTF and ICD in OPCs (Sakry et al., 2014Sakry and Trotter, 2016). ...
The NG2 proteoglycan is expressed by oligodendrocyte precursor cells (OPCs) and is abundantly expressed by tumors such as melanoma and glioblastoma. Functions of NG2 include an influence on proliferation, migration and neuromodulation. Similar to other type-1 membrane proteins, NG2 undergoes proteolysis, generating a large ectodomain, a C-terminal fragment (CTF) and an intracellular domain (ICD) via sequential action of α- and γ-secretases which is enhanced by neuronal activity. Functional roles of NG2 have so far been shown for the full-length protein, the released ectodomain and CTF, but not for the ICD. In this study, we characterized the role of the NG2 ICD in OPC and Human Embryonic Kidney (HEK) cells. Overexpressed ICD is predominantly localized in the cell cytosol, including the distal processes of OPCs. Nuclear localisation of a fraction of the ICD is dependent on Nuclear Localisation Signals. Immunoprecipitation and Mass Spectrometry followed by functional analysis indicated that the NG2 ICD modulates mRNA translation and cell-cycle kinetics. In OPCs and HEK cells, ICD overexpression results in an mTORC1-dependent upregulation of translation, as well as a shift of the cell population toward S-phase. NG2 ICD increases the active (phosphorylated) form of mTOR and modulates downstream signaling cascades, including increased phosphorylation of p70S6K1 and increased expression of eEF2. Strikingly, levels of FMRP, an RNA-binding protein that is regulated by mTOR/p70S6K1/eEF2 were decreased. In neurons, FMRP acts as a translational repressor under activity-dependent control and is mutated in Fragile X Syndrome (FXS). Knock-down of endogenous NG2 in primary OPC reduced translation and mTOR/p70S6K1 phosphorylation in Oli-neu. Here, we identify the NG2 ICD as a regulator of translation in OPCs via modulation of the well-established mTORC1 pathway. We show that FXS-related FMRP signaling is not exclusive to neurons but plays a role in OPCs. This provides a signal cascade in OPC which can be influenced by the neuronal network, since the NG2 ICD has been shown to be generated by constitutive as well as activity-dependent cleavage. Our results also elucidate a possible role of NG2 in tumors exhibiting enhanced rates of translation and rapid cell cycle kinetics.
... Indeed, proteins implicated in early-and late-onset neurodegeneration are massively expressed throughout embryogenesis and frequently impact on different steps of neural development, such as neuronal proliferation, differentiation and migration, axon targeting or synaptogenesis. Furthermore, these affected molecular bases underlying belated neurodegenerative processes are often embedded into key developmental signalling pathways in both invertebrate and vertebrate pathogenic models, such as the Notch (Levitan and Greenwald, 1995) and Wnt (Nishimura et al., 1999) pathways in Alzheimer's disease, Wnt signalling in Huntington disease (Dupont et al., 2012;Tourette et al., 2014) or the bone morphogenetic protein (BMP) pathway in motor neurodegeneration (Wang et al., 2007;Chang et al., 2008;Ratnaparkhi et al., 2008;Fassier et al., 2010;Nahm et al., 2013). ...
Functional analyses of genes responsible for neurodegenerative disorders have unveiled critical links between neurodegenerative processes and key developmental signalling pathways. Mutations in SPG4 encoding spastin cause hereditary spastic paraplegia (HSP). Spastin is involved in diverse cellular processes coupling microtubule severing to membrane remodelling. Two main spastin isoforms are synthesised from alternative translational start sites (M1 and M87). However, their specific roles in neuronal development and homeostasis remain largely unknown. To selectively unravel their neuronal function, we blocked Spastin synthesis from each initiation codon during zebrafish development and performed rescue analyses. The knockdown of each isoform led to different motoneuron and locomotion defects, which were not rescued by the selective expression of the other isoform. Notably, both morphant neuronal phenotypes were observed in a CRISPR/Cas9 spastin mutant. We next show that M1 spastin together with HSP proteins atlastin-1 and NIPA1 drive motor axon targeting by repressing BMP signalling, while M87 spastin acts downstream Neuropilin-1 to control motoneuron migration. Our data thus suggest that defective BMP and Neuropilin-1 signalling may contribute to the motor phenotype in a vertebrate model of spastin depletion.
Alzheimer's disease (AD) is typified by memory deficiencies that worsen with time, along with cognitive and behavioral abnormalities that eventually result in dementia. AD is one of the most difficult diseases to treat because of its prevalence, high cost of care, effects on patients and caregivers, and lack of mechanism-based therapies. The malfunction and loss of neurons in particular circuits and locations, especially in the populations of nerve cells supporting memory and cognition, causes the AD condition.The neuropathology of AD is characterized by intracellular and extracellular protein aggregation accumulations. In the neuronal perikarya and dystrophic neurites, abnormally phosphorylated tau forms paired helical filaments (PHFs) that combine to form neurofibrillary tangles (NFTs). The extracellular deposition of β-pleated assemblies of Aβ peptide, which result in widespread and neuritic senile plaques, is the second pathogenic hallmark.
Objective
Mutations in presenilin genes are the major cause of Alzheimer’s disease. However, little is known about their expression and function in the gut. In this study, we identify the presenilins Psen1 and Psen2 as key molecules that maintain intestinal homoeostasis.
Design
Human inflammatory bowel disease (IBD) and control samples were analysed for Psen1 expression. Newly generated intestinal epithelium-specific Psen1-deficient, Psen2-deficient and inducible Psen1/Psen2 double-deficient mice were used to dissect the functional role of presenilins in intestinal homoeostasis.
Results
Psen1 expression was regulated in experimental gut inflammation and in patients with IBD. Induced deletion of Psen1 and Psen2 in mice caused rapid weight loss and spontaneous development of intestinal inflammation. Mice exhibited epithelial barrier disruption with bacterial translocation and deregulation of key pathways for nutrient uptake. Wasting disease was independent of gut inflammation and dysbiosis, as depletion of microbiota rescued Psen-deficient animals from spontaneous colitis development but not from weight loss. On a molecular level, intestinal epithelial cells lacking Psen showed impaired Notch signalling and dysregulated epithelial differentiation.
Conclusion
Overall, our study provides evidence that Psen1 and Psen2 are important guardians of intestinal homoeostasis and future targets for barrier-promoting therapeutic strategies in IBD.
Two phase-III clinical trials with anti-amyloid peptide antibodies have met their primary goal, i.e. slowing of Alzheimer’s disease (AD) progression. However, antibody therapy may not be the optimal therapeutic modality for AD prevention, as we will discuss in the context of the earlier small molecules described as “γ-secretase modulators” (GSM). We review here the structure, function, and pathobiology of γ-secretases, with a focus on how mutations in presenilin genes result in early-onset AD. Significant progress has been made in generating compounds that act in a manner opposite to pathogenic presenilin mutations: they stabilize the proteinase-substrate complex, thereby increasing the processivity of substrate cleavage and altering the size spectrum of Aβ peptides produced. We propose the term “γ-secretase allosteric stabilizers” (GSAS) to distinguish these compounds from the rather heterogenous class of GSM. The GSAS represent, in theory, a precision medicine approach to the prevention of amyloid deposition, as they specifically target a discrete aspect in a complex cell biological signalling mechanism that initiates the pathological processes leading to Alzheimer’s disease.
The γ-secretase complexes are intramembrane cleaving proteases involved in the generation of the Aβ peptides in Alzheimer’s disease. The complex consists of four subunits, with Presenilin harboring the catalytic site. Here, we study the role of the smallest subunit, PSENEN or Presenilin enhancer 2, encoded by the gene Psenen, in vivo and in vitro. We find a profound Notch deficiency phenotype in Psenen−/− embryos confirming the essential role of PSENEN in the γ-secretase complex. We used Psenen−/− fibroblasts to explore the structure–function of PSENEN by the scanning cysteine accessibility method. Glycine 22 and proline 27, which border the membrane domains 1 and 2 of PSENEN, are involved in complex formation and stabilization of γ-secretase. The hairpin structured hydrophobic membrane domains 1 and 2 are exposed to a water-containing cavity in the complex, while transmembrane domain 3 is not water exposed. We finally demonstrate the essential role of PSENEN for the cleavage activity of the complex. PSENEN is more than a structural component of the γ-secretase complex and might contribute to the catalytic mechanism of the enzyme.
Caenorhabditis elegans is an exceptional model organism. More than twenty thousand different strains have been developed, increasing knowledge on countless topics. However, the traditional method to cryopreserve this nematode, based on slow freezing, usually reaches recovery rates of around 35% for the L1 and L2 larval stages. Here, we propose two alternative methods to cryopreserve this nematode based on vitrification that are applicable in common laboratories and allow the selective individual cryopreservation of this organism. These new methods require ultra-high warming rates, which are achieved by employing very thin capillaries as the nematode container, and a very low final concentration of cryoprotectants, which, as compared to slow freezing, reduce toxicity damage. The recovery rate was 98.5% for larvae (L1 - L4) and 84.3% for adults. Given these results, our procedures offer an alternative to cryopreserve this nematode (larvae and adults) with higher recovery rates, avoiding expensive requirements. Indeed, it only needed a container with liquid nitrogen and a warming bath for water at 37 °C. The high performance of this approach has been revealed by preserving the long-term memory and, probably, the connectome of this nematode.
The gamma secretase complexes are intramembrane cleaving proteases involved in the generation of the Abeta peptides in Alzheimers Disease. The complex consists of four subunits, with Presenilin, harboring the catalytic site. Here, we study the role of the smallest subunit, PSENEN or Presenilin enhancer 2 (PEN2), encoded by the gene Psenen, in vivo and in vitro. We find a profound Notch deficiency phenotype in Psenen knockout embryos confirming the essential role of PSENEN in the gamma-secretase complex. We used Psenen knockout fibroblasts to explore the structure function of PSENEN by the Scanning Cysteine Accessibility Method. Glycine22 and Proline27 which border the membrane domains 1 and 2 of PSENEN are involved in complex formation and stabilization of gamma secretase. The hairpin structured hydrophobic membrane domains 1 and 2 are exposed to a water containing cavity in the complex, while transmembrane domain 3 is not water exposed. We finally demonstrate the essential role of PSENEN for the cleavage activity of the complex. PSENEN is more than a structural component of the gamma secretase complex and might contribute to the catalytic mechanism of the enzyme.
Mutations in the presenilin (PS) genes are a predominant cause of familial Alzheimer’s disease (fAD). An ortholog of PS in the genetic model organism Caenorhabditis elegans (C. elegans) is sel-12. Mutations in the presenilin genes are commonly thought to lead to fAD by upregulating the expression of amyloid beta (Ab), however this hypothesis has been challenged by recent evidence. As C. elegans lack amyloid beta (Ab), the goal of this work was to examine Ab-independent effects of mutations in sel-12 and PS1/PS2 on behaviour and sensory neuron morphology across the lifespan in a C. elegans model. Olfactory chemotaxis experiments were conducted on sel-12(ok2078) loss-of-function mutant worms. Adult sel-12 mutant worms showed significantly lower levels of chemotaxis to odorants compared to wild-type worms throughout their lifespan, and this deficit increased with age. The chemotaxis phenotype in sel-12 mutant worms is rescued by transgenic over-expression of human wild-type PS1, but not the classic fAD-associated variant PS1C410Y, when expression was driven by either the endogenous sel-12 promoter (Psel-12), a pan-neuronal promoter (Primb-1), or by a promoter whose primary expression was in the sensory neurons responsible for the chemotaxis behavior (Psra-6, Podr-10). The behavioural phenotype was also rescued by over-expressing an atypical fAD-linked mutation in PS1 (PS1?S169) that has been reported to leave the Notch pathway intact. An examination of the morphology of polymodal nocieptive (ASH) neurons responsible for the chemotaxis behavior also showed increased neurodegeneration over time in sel-12 mutant worms that could be rescued by the same transgenes that rescued the behaviour, demonstrating a parallel with the observed behavioral deficits. Thus, we report an Ab-independent neurodegeneration in C. elegans that was rescued by cell specific over-expression of wild-type human presenilin.
A genetic study of familial cases of Alzheimer’s disease (AD) has revealed that amyloid-β protein (Aβ) is an important pathogenic protein in AD. Aβ is derived from its precursor protein APP by sequential cleavages mediated by β- and γ-secretases. Presenilin (PS), which is one of the major familial AD-linked genes, is a catalytic subunit of the γ-secretase. γ-Secretase is an atypical protease complex composed of four membrane proteins; PS, nicastrin, anterior pharyngeal defective 1 (Aph-1), and presenilin enhancer 2 (Pen-2). Familial AD-linked genetic mutations in PS genes augment the production of the longer form of Aβ, which is an aggregation-prone peptide. However, the development of inhibitors for γ-secretase has been discontinued so far due to side effects. In contrast, the studies of γ-secretase led to new enzymology of intramembrane proteolysis of the transmembrane domains. Furthermore, a novel class of compounds called γ-secretase modulators has been developed. In this article, we discuss the current understanding of the basic and therapeutic aspects of PS/γ-secretase.KeywordsAlzheimer’s disease, Amyloid-β, γ-Secretase, Intramembrane proteolysis, Presenilin, Protease
Computational methods and tools are a powerful complementary approach to experimental work for studying regulatory interactions in living cells and systems. We demonstrate the use of formal reasoning methods as applied to the Caenorhabditis elegans germ line, which is an accessible system for stem cell research. The dynamics of the underlying genetic networks and their potential regulatory interactions are key for understanding mechanisms that control cellular decision-making between stem cells and differentiation.We model the “stem cell fate” versus entry into the “meiotic development” pathway decision circuit in the young adult germ line based on an extensive study of published experimental data and known/hypothesized genetic interactions. We apply a formal reasoning framework to derive predictive networks for control of differentiation. Using this approach we simultaneously specify many possible scenarios and experiments together with potential genetic interactions, and synthesize genetic networks consistent with all encoded experimental observations. In silico analysis of knock-down and overexpression experiments within our model recapitulate published phenotypes of mutant animals and can be applied to make predictions on cellular decision-making. A methodological contribution of this work is demonstrating how to effectively model within a formal reasoning framework a complex genetic network with a wealth of known experimental data and constraints. We provide a summary of the steps we have found useful for the development and analysis of this model and can potentially be applicable to other genetic networks. This work also lays a foundation for developing realistic whole tissue models of the C. elegans germ line where each cell in the model will execute a synthesized genetic network.
Several alternative models have been used to create new studies on central nervous system (CNS) function. The benefits associated with these alternative approaches are time efficiency, reduced manpower, cost-effectiveness, and avoidance of ethical concerns on the use of higher vertebrates. In this regard, Caenorhabditis elegans (C. elegans), a small, free-living, soil nematode which uses bacteria as a food source, is being used to complement traditional experiments. Studies on worm genomes add new insights into the mechanisms of several neurological disorders and neuronal regeneration. Strains can be genetically modified to express fluorescent reporters, allowing one to follow relevant regulatory pathways, visualize different neuronal lineages as well as morphology alterations. One key advantage of studies with C. elegans is that a functional synapse can be studied inside a living organism and an immediate behavioral response profile can be observed. Neurotoxicology studies using C. elegans allow the discovery of a collection of genes and molecular targets involved in neurodegeneration and other neuronal alterations following toxicological insult. Here we provide a synopsis of the findings on the cholinergic and dopaminergic systems of the CNS, using C. elegans as an animal model.KeywordsDopamineDopaminergic systemAcetylcholineCholinergic systemNeurodegenerationParkinson disease
Computational methods and tools are a powerful complementary approach to experimental work for studying regulatory interactions in living cells and systems. We demonstrate the use of formal reasoning methods as applied to the Caenorhabditis elegans germ line, which is an accessible model system for stem cell research. The dynamics of the underlying genetic networks and their potential regulatory interactions are key for understanding mechanisms that control cellular decision-making between stem cells and differentiation.We model the "stem cell fate" versus entry into the "meiotic development" pathway decision circuit in the young adult germ line based on an extensive study of published experimental data and known/hypothesized genetic interactions. We apply a formal reasoning framework to derive predictive networks for control of differentiation. Using this approach we simultaneously specify many possible scenarios and experiments together with potential genetic interactions, and synthesize genetic networks consistent with all encoded experimental observations. In silico analysis of knock-down and overexpression experiments within our model recapitulate published phenotypes of mutant animals and can be applied to make predictions on cellular decision-making. This work lays a foundation for developing realistic whole tissue models of the C. elegans germ line where each cell in the model will execute a synthesized genetic network.
Ligands present on neighboring cells activate receptors of the LIN-12/Notch family by inducing a proteolytic cleavage event that releases the intracellular domain. Mutations that appear to eliminate sel-5 activity are able to suppress constitutive activity of lin-12(d) mutations that are point mutations in the extracellular domain of LIN-12, but cannot suppress lin-12(intra), the untethered intracellular domain. These results suggest that sel-5 acts prior to or during ligand-dependent release of the intracellular domain. In addition, sel-5 suppression of lin-12(d) mutations is tissue specific: loss of sel-5 activity can suppress defects in the anchor cell/ventral uterine precursor cell fate decision and a sex myoblast/coelomocyte decision, but cannot suppress defects in two different ventral hypodermal cell fate decisions in hermaphrodites and males. sel-5 encodes at least two proteins, from alternatively spliced mRNAs, that share an amino-terminal region and differ in the carboxy-terminal region. The amino-terminal region contains the hallmarks of a serine/threonine kinase domain, which is most similar to mammalian GAK1 and yeast Pak1p.
Caenorhabditis elegans (C. elegans) as a well-established multicellular model organism has been widely used in the biological field for half a century. Its numerous advantages including small body size, rapid life cycle, high-reproductive rate, well-defined anatomy, and conserved genome, has made C. elegans one of the most successful multicellular model organisms. Discoveries obtained from the C. elegans model have made great contributions to research fields such as development, aging, biophysics, immunology, and neuroscience. Because of its transparent body and giant cell size, C. elegans is also an ideal subject for high resolution and high-throughput optical imaging and analysis. During the past decade, great advances have been made to develop biomolecule-targeting techniques for noninvasive optical imaging. These novel technologies expanded the toolbox for qualitative and quantitative analysis of biomolecules in C. elegans. In this review, we summarize recently developed fluorescent probes or labeling techniques for visualizing biomolecules at the cellular, subcellular or molecular scale by using C. elegans as the major model organism or designed specifically for the applications in C. elegans. Combining the technological advantages of the C. elegans model with the novel fluorescent labeling techniques will provide new horizons for high-efficiency quantitative optical analysis in live organisms.
The conduction of the XIX Symposium of the International Association for Comparative Research on Leukemia and Related Diseases (IACRLRD) led the authors to write an article introducing comparative research in leukemia and related diseases. Our survey briefly summarizes the history of this symposium, as it evolved from a meeting on animal leukemia viruses into one dealing with viral and genetic aspects of human and animal leukemia and related diseases including AIDS. The scientific evolution of the Abelson murine leukemia virus with its abl oncogene in the 1970s, to what currently appears as the most reliable marker for human chronic myeloid leukemia (CML), is just one example.
How multiple intercellular signals are integrated by cells to generate a specific pattern of cell fates in a reproducible manner is the key to understand the development of multicellular organisms. Vulval development has proved to be an excellent model to study this question at the molecular level. Furthermore, since the specification of the vulval cell fates occurs in a set of epithelial precursor cells, the specific requirements for signaling processes to occur in polarized, asymmetric cells can be investigated. Cell fate determination during vulval development is thus likely to be generally applicable to more complex organisms. The vulva is formed by the 22 descendants of P5.p, P6.p, and P7.p, three of the 12 epithelial Pn.p cells that are positioned along the ventral midline of the animal. During wild-type vulval development, the anchor cell in the gonad induces P6.p to go through three rounds of symmetric cell divisions and generate 8 descendants—a lineage termed the primary (1°) cell fate. In response to the inductive anchor cell signal, P6.p sends a lateral signal that induces the secondary (2°) cell fate in the two adjacent P5.p and P7.p and prevents them from adopting the 1° cell fate.
Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.
The Notch pathway controls a very broad spectrum of cell fates in metazoans during development, influencing proliferation, differentiation and cell death. Given its central role in normal development and homeostasis, misregulation of Notch signals can lead to various disorders including cancer. How the Notch pathway mediates such pleiotropic and differential effects is of fundamental importance. It is becoming increasingly clear through a number of large-scale genetic and proteomic studies that Notch interacts with a staggeringly large number of other genes and pathways in a context-dependent, complex, and highly regulated network, which determines the ultimate biological outcome. How best to interpret and analyze the continuously increasing wealth of data on Notch interactors remains a challenge. Here we review the current state of genetic and proteomic data related to the Notch interactome.
We have performed single pass sequencing of 1,024 human brain cDNAs, over 900 of which seem to represent new human genes. Library prescreening with total brain cDNA significantly reduced repeated sequencing of highly represented cDNAs. A subset of sequenced cDNAs were physically mapped to their chromosomal locations using gene-specific STS primers derived from 3' untranslated regions. We have also determined that human brain cDNAs represent a rich source of gene-associated polymorphic markers. Microsatellite-containing cDNAs can be physically mapped and converted to highly informative genetic markers, thus facilitating integration of the human physical, expression and genetic maps.
Spermatogenesis in the nematode Caenorhabditis elegans uses unusual organelles, called the fibrous body-membranous organelle (FB-MO) complexes, to prepackage and deliver macromolecules to spermatids during cytokinesis that accompanies the second meiotic division. Mutations in the spe-4 (spermatogenesis-defective) gene disrupt these organelles and prevent cytokinesis during spermatogenesis, but do not prevent completion of the meiotic nuclear divisions that normally accompany spermatid formation. We report an ultrastructural analysis of spe-4 mutant sperm where the normally close association of the FB's with the MO's and the double layered membrane surrounding the FB's are both defective. The internal membrane structure of the MO's is also disrupted in spe-4 mutant sperm. Although sperm morphogenesis in spe-4 mutants arrests prior to the formation of spermatids, meiosis can apparently be completed so that haploid nuclei reside in an arrested spermatocyte. We have cloned the spe-4 gene in order to understand its role during spermatogenesis and the molecular basis of how mutation of this gene disrupts this process. The spe-4 gene encodes an approximately 1.5-kb mRNA that is expressed during spermatogenesis, and the sequence of this gene suggests that it encodes an integral membrane protein. These data suggest that mutation of an integral membrane protein within FB-MO complexes disrupts morphogenesis and prevents formation of spermatids but does not affect completion of the meiotic nuclear divisions in C. elegans sperm.
Two homologous genes, lin-12 and glp-1, encode transmembrane proteins required for regulatory cell interactions during C. elegans development. Based on their single mutant phenotypes, each gene has been thought to govern a distinct set of cell fates. We show here that lin-12 and glp-1 are functionally redundant during embryogenesis: Unlike either single mutant, the lin-12 glp-1 double mutant dies soon after hatching. Numerous cellular defects can be observed in these Lag (for lin-12 and glp-1) double mutants. Furthermore, we have identified two genes, lag-1 and lag-2, that appear to be required for both lin-12 and glp-1-mediated cell interactions. Strong loss-of-function lag mutants are phenotypically indistinguishable from the lin-12 glp-1 double; weak lag mutants have phenotypes typical of lin-12 and glp-1 single mutants. We speculate that the lin-12 and glp-1 proteins are biochemically interchangeable and that their divergent roles in development may rely largely on differences in gene expression.
We report a rapid technique, based on the polymerase chain reaction (PCR), for the direct targeting, enhancement, and sequencing of previously uncharacterized cDNAs. This method is not limited to previously sequenced transcripts, since it requires only two adjacent or partially overlapping specific primers from only one side of the region to be amplified. These primers can be located anywhere within the message. The specific primers are used in conjunction with nonspecific primers targeted either to the poly(A)+ region of the message or to an enzymatically synthesized d(A) tail. Pairwise combinations of specific and general primers allow for the amplification of regions both 3' and 5' to the point of entry into the message. The amplified PCR products can be cloned, sequenced directly by genomic sequencing, or labeled for sequencing by amplifying with a radioactive primer. We illustrate the power of this approach by deriving the cDNA sequences for the skeletal muscle alpha-tropomyosins of European common frog (Rana temporaria) and zebrafish (Brachydanio rerio) using only 300 ng of a total poly(A)+ preparation. In these examples, we gained initial entry into the tropomyosin messages by using heterologous primers (to conserved regions) derived from the rat skeletal muscle alpha-tropomyosin sequence. The frog and zebrafish sequences are used in an analysis of tropomyosin evolution across the vertebrate phylogenetic spectrum. The results underscore the conservative nature of the tropomyosin molecule and support the notion of a constrained heptapeptide unit as the fundamental structural motif of tropomyosin.
We describe a dominant behavioral marker, rol‐6(su‐1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double‐strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single‐stranded oligonucleotide was co‐injected with the double‐stranded DNA.
The role of cell-cell interaction in the postembryonic development of nongonadal tissues in the nematode Caenorhabditis elegans has been explored by selective cell ablation with a laser microbeam. Examples have been found of induction and of regulation in cell lineage and fate. Regulation in which one cell precisely or partially replaces another is seen, but only in certain groups of hypodermal cells which resemble one another closely; cells which are unique are not replaced in this way. The regulation of cell form is more widespread and less restrictive.
The number of nongonadal nuclei in the free-living soil nematode Caenorhabditis elegans increases from about 550 in the newly hatched larva to about 810 in the mature hermaphrodite and to about 970 in the mature male. The pattern of cell divisions which leads to this increase is essentially invariant among individuals; rigidly determined cell lineages generate a fixed number of progeny cells of strictly specified fates. These lineages range in length from one to eight sequential divisions and lead to significant developmental changes in the neuronal, muscular, hypodermal, and digestive systems. Frequently, several blast cells follow the same asymmetric program of divisions; lineally equivalent progeny of such cells generally differentiate into functionally equivalent cells. We have determined these cell lineages by direct observation of the divisions, migrations, and deaths of individual cells in living nematodes. Many of the cell lineages are involved in sexual maturation. At hatching, the hermaphrodite and male are almost identical morphologically; by the adult stage, gross anatomical differences are obvious. Some of these sexual differences arise from blast cells whose division patterns are initially identical in the male and in the hermaphrodite but later diverge. In the hermaphrodite, these cells produce structures used in egg-laying and mating, whereas, in the male, they produce morphologically different structures which function before and during copulation. In addition, development of the male involves a number of lineages derived from cells which do not divide in the hermaphrodite. Similar postembryonic developmental events occur in other nematode species.
Developmental, genetic and molecular studies indicate that multiple intercellular signalling systems interact to specify the types and spatial patterns of cells generated during the formation of the vulva of the nematode Caenorhabditis elegans. Two classes of evolutionarily conserved transmembrane receptors and a Ras protein function in these signalling systems. The biology of vulval development provides a framework for understanding how cell interactions control the development of animals as diverse as nematodes, insects and mammals.
The let-60 gene, an essential ras gene of the nematode Caenorhabditis elegans, acts as a switch in the inductive signalling pathway that initiates vulva formation. Recessive let-60 mutations that cause a vulvaless phenotype prevent let-60 function in response to the inductive signal. These mutations are clustered and define regions necessary either for the activation or for the action of the let-60 ras protein. Dominant let-60 mutations that cause a multivulva phenotype alter codon 13 and activate let-60 in vivo, rendering it independent of the inductive signal. The let-60 gene acts within an extensively defined genetic pathway, and other genes within this pathway seem likely to encode molecules that regulate let-60 function as well as molecules that are targets of let-60 action.
Certain cell fate decisions are specified by cell-cell interactions during the development of the nematode Caenorhabditis elegans. For example, in a wild-type hermaphrodite gonad, two cells, Z1.ppp and Z4.aaa, have the potential to become the anchor cell (AC). Intercellular communication establishes their fates and ensures that only one cell becomes the AC, while the other becomes a ventral uterine precursor cell (VU). One component of this intercellular communication seems to be the 'AC-to-VU' signal from the presumptive AC that causes the other cell to become a VU. Genetic and developmental studies indicated that the lin-12 gene specifies the fates of Z1.ppp and Z4.aaa. Molecular studies suggest that lin-12 directly participates in their communications, perhaps acting as the receptor for the 'AC-to-VU' signal. Here, we report the molecular lesions associated with lin-12 gain-of-function mutations, cell isolation experiments, and genetic studies of an unusual lin-12 allele. These data suggest that self-association of the putative lin-12-encoded receptor leads to its activation, and that certain gain-of-function mutations result in ligand-independent activation.
Genetic analysis previously suggested that the let-60 gene controls the switch between vulval and hypodermal cell fates during C. elegans vulval induction. We have cloned the let-60 gene, and shown that it encodes a gene product identical in 84% of its first 164 amino acids to ras gene products from other vertebrate and invertebrate species. This conservation suggests that the let-60 product contains all the biochemical functions of ras proteins. Extrachromosomal arrays of let-60 ras DNA cause cell-type misspecification (extra vulval fates) phenotypically opposite to that caused by let-60 ras loss-of-function mutations (no vulval fates), and suppress the vulvaless phenotype of mutations in two other genes necessary for vulval induction. Thus, the level and pattern of let-60 ras expression may be under strict regulation; increase in let-60 ras activity bypasses or reduces the need for upstream genes in the vulval induction pathway.
Each of the six C. elegans vulval precursor cells (VPCs) has three potential fates (1 degree, 2 degrees, or 3 degrees). The fate of each VPC depends on two types of signals: a graded inductive signal that acts at a distance and a short-range lateral signal among the VPCs. We describe interactions among mutations that cause different misspecifications of VPC fates. Particular combinations of mutations cause all six VPCs to have a single fate independent of their positions. Our results suggest that specification of the three VPC fates is accomplished by two binary decisions, each effected by one of the two signaling pathways. The gene lin-12 acts in the lateral signaling pathway and specifies 2 degrees. The "vulvaless" and "multivulva" genes act in the inductive signaling pathway and specify 1 degree independently of lin-12 and 2 degrees via lin-12. We describe a model for the regulatory circuitry underlying VPC determination that includes a role for lin-12 in both autocrine and paracrine VPC signaling.
The lin-12 gene of C. elegans encodes a predicted transmembrane protein that controls a decision by two cells, Z1.ppp and Z4.aaa, between the anchor cell (AC) and ventral uterine precursor cell (VU) fates. We performed laser ablation experiments to demonstrate that specification of the VU fate of Z1.ppp or Z4.aaa depends on an "AC-to-VU" signal from the presumptive AC. We generated genetic mosaics in which defined cells lacked lin-12 activity. By correlating the fates of Z1.ppp and Z4.aaa with the lin-12 genotype of nearly every cell in these mosaics, we conclude that lin-12 function is VU cell autonomous. We present a model in which lin-12 functions in the receiving mechanism for the "AC-to-VU" signal leading to the specification of the AC and VU fates of Z1.ppp and Z4.aaa.
The haploid genome of Caenorhabditis elegans consists of some 80 x 10(6) base pairs of DNA contained in six chromosomes. The large number of interesting loci that have been recognized by mutation, and the accuracy of the genetic map, mean that a physical map of the genome is highly desirable, because it will facilitate the molecular cloning of chosen loci. The first steps towards such a map used a fingerprinting method to link cosmid clones together. This approach reached its practical limit last year, when 90-95% of the genome had been cloned into 17,500 cosmids assembled into some 700 clusters (contigs), but the linking clones needed were either non-existent or extremely rare. Anticipating this, we had planned to link by physical means--probably by hybridization to NotI fragments separated by pulse field gel electrophoresis. NotI recognizes an eight base sequence of GC pairs; thus the fragments should be large enough to bridge regions that clone poorly in cosmids, and, with no selective step involved, would necessarily be fully representative. However, with the availability of a yeast artificial chromosome (YAC) vector, we decided to use this alternative source of large DNA fragments to obtain linkage. The technique involves the ligation of large (50-1,000 kilobase) genomic fragments into a vector that provides centromeric, telomeric and selective functions; the constructs are then introduced into Saccharomyces cerevisiae, and replicate in the same manner as the host chromosomes.
During Caenorhabditis elegans vulval induction the anchor cell of the gonad specifies a spatial pattern of three cell types among a set of six multipotent epidermal cells, the vulval precursor cells (VPCs). Previous studies suggested that the anchor cell produces a graded inductive signal which can directly stimulate VPCs away from a ground state (type 3) to become type 1 or type 2 depending on their distance from the anchor cell. Here, we investigate the interactions among VPCs in a mutant, lin-15, in which VPC fates are rendered partially independent of the inductive signal, and show that type 1 cells actively inhibit adjacent cells from also becoming type 1 cells. The fate of each VPC therefore depends on the combined action of two intercellular signals: a graded inductive signal from the anchor cell, and a lateral inhibitory signal from at least some of its neighbours. Pattern formation among the VPCs lin-15 mutant is analogous to the establishment of the pattern of neuroblasts and dermatoblasts during early insect neurogenesis, suggesting that the similarities in inferred molecular structure of the lin-12 and Notch gene products, which are involved in these two instances of pattern formation, might extend to similarities in function.
While determining the 5' ends of C. elegans actin mRNAs, we have discovered a 22 nucleotide spliced leader sequence. The leader sequence is found on mRNA from three of the four nematode actin genes. The leader also appears to be present on some, but not all, nonactin mRNAs. The actin mRNA leader sequence is identical to the first 22 nucleotides of a novel 100 nucleotide RNA transcribed adjacent, and in the opposite orientation, to the 5S ribosomal gene. The evidence suggests that the actin mRNA leader sequence is acquired from this novel nucleotide transcript by an intermolecular trans-splicing mechanism.
Interactions between the early blastomeres in a C. elegans embryo are required for the specification of certain cell fates. Blastomeres that produce neurons and skin cells when cultured in isolation are induced to also produce pharyngeal cells in intact embryos. We have identified maternal effect lethal mutations that, on the basis of phenotype and temperature-sensitive period, appear to disrupt this inductive interaction. These mutations are all alleles of glp-1, a gene also involved in the control of germ cell proliferation during postembryonic development of C. elegans.
In the wild-type C. elegans germ line there are both mitotic and meiotic germ cells. Mutations in glp-1 cause germ cells that would normally divide mitotically to enter meiosis. This mutant phenotype mimics the effect of killing the distal tip cell, a somatic cell that interacts with the germ line to regulate the mitotic/meiotic decision. In addition, wild-type glp-1 product is required maternally for embryogenesis. Temperature-shift experiments indicate that the temporal requirement for glp-1 activity in the germ line is the same as that for distal tip cell regulation. Mosaic analyses suggest that glp-1 is produced in the germ line. We propose that glp-1 acts as part of the receiving mechanism in the interaction between the distal tip cell and germ line.
Previous studies have shown that the development of the vulva of the C. elegans hermaphrodite involves six multipotential hypodermal cells as well as the gonadal anchor cell, which induces vulval formation. Our further examination of the interactions among these seven cells has led to the following model. Each hypodermal precursor cell becomes determined to adopt one of its three potential fates; each of these fates is to generate a particular cell lineage. In the absence of cellular interactions each precursor cell will generate the nonvulval cell lineage; an inductive signal from the anchor cell is required for a precursor cell to generate either of the two types of vulval cell lineages. The inductive signal is spatially graded, and the potency of the signal specifies which lineage is expressed by each of the tripotential precursor cells.
Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C. elegans are large.
We describe two classes of mutations in the lin-12 locus of the nematode Caenorhabditis elegans. Ten semidominant mutations (lin-12[d]) appear to elevate the level of lin-12 activity. Thirty-two recessive alleles (lin-12[0]), including two amber mutations, appear to eliminate gene activity. The lin-12(d) and lin-12(0) mutations result in reciprocal homeotic transformations in the fates of defined cells in several different tissues. Gene dosage studies suggest that a high level of lin-12 activity specifies one cell fate and a low level specifies an alternative fate. Temperature-shift experiments indicate that lin-12 acts at the time cell fate is determined in wild type. We propose that lin-12 functions as a binary switch to control decisions between alternative cell fates during C. elegans development.
After hatching, the germ line progenitor cells in C. elegans begin to divide mitotically; later, some of the germ line cells enter meiosis and differentiate into gametes. In the adult, mitotic germ cells, or stem cells, are found at one end (the distal end) and meiotic cells occupy the rest of the elongate gonad. Removal of two somatic gonadal cells, the distal tip cells, by laser microsurgery has a dramatic effect on germ cell development. In either sex, this operation leads to the arrest of mitosis and the initiation of meiosis in germ cells. The function of the distal tip cell in the intact animal appears to be the inhibition of meiosis (or stimulation of mitosis) in nearby germ cells. During development, this permits growth and, in the adult, it maintains the germ line stem cell population. A change in the position of the distal tip cell in the gonad at an early point in development is correlated with a change in the axial polarity of the germ line tissue. This suggests that the localization of the distal tip cell's inhibitory activity at the distal end of the gonad establishes the axial polarity of the germ line tissue in the intact animal.
The postembryonic cell lineage of the somatic gonad is essentially invariant in Caenorhabditis elegans (J.E. Kimble and D. Hirsh, 1979, Develop. Biol.70, 396–417). The two exceptions to this rule of invariance involve a natural ambiguity in the ancestry of certain cells such that each of two precursor cells assumes one of two alternative fates in a given animal. In this paper, experiments are reported in which laser microsurgery is used to kill individual cells in the developing somatic gonad. Such intervention perturbs the normal environment of the remaining cells; a change observed in the expected behavior of these cells suggests that extrinsic cues may normally play a role in controlling that behavior. Several different lineage alterations have been observed after laser microsurgery in the somatic gonad. These include switches in the type of lineage followed by a given precursor cell, reversals in lineage polarity, duplications of a lineage, and alteratiions in the number of cells produced in the lineage. The only cases in which cells switch from one lineage type to another involve pairs of cells which exhibit natural ambiguity. In most cases, the interactions inferred from these changes seem to occur between neighboring somatic gonadal cells. In one case, induction of the vulva, the interaction occurs between a single somatic gonadal cell, the anchor cell, and the precursors to the vulva in a neighboring tissue, the hypodermis. The roles of intrinsic and extrinsic cues in controlling normally invariant cell lineages are discussed.
Some cases of Alzheimer's disease are inherited as an autosomal dominant trait. Genetic linkage studies have mapped a locus (AD3) associated with susceptibility to a very aggressive form of Alzheimer's disease to chromosome 14q24.3. We have defined a minimal cosegregating region containing the AD3 gene, and isolated at least 19 different transcripts encoded within this region. One of these transcripts (S182) corresponds to a novel gene whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein. Five different missense mutations have been found that cosegregate with early-onset familial Alzheimer's disease. Because these changes occurred in conserved domains of this gene, and are not present in normal controls, they are likely to be causative of AD3.
The Notch/Lin-12/Glp-1 receptor family mediates the specification of numerous cell fates during development in Drosophila
and Caenorhabditis elegans. Studies on the expression, mutant phenotypes, and developmental consequences of unregulated receptor
activation have implicated these proteins in a general mechanism of local cell signaling, which includes interactions between
equivalent cells and between different cell types. Genetic approaches in flies and worms have identified putative components
of the signaling cascade, including a conserved family of extracellular ligands and two cellular factors that may associate
with the Notch Intracellular domain. One factor, the Drosophila Suppressor of Hairless protein, is a DNA-binding protein,
which suggests that Notch signaling may involve relatively direct signal transmission from the cell surface to the nucleus.
Several vertebrate Notch receptors have also been discovered recently and play important roles in normal development and tumorigenesis.
During the induction of the Caenorhabditis elegans vulva, cell signalling causes initially equipotent cells to express a reproducible pattern of cell fates. The position of the anchor cell determines the pattern of vulval precursor cell fates, such that the closest precursor cell (P6.p) expresses the primary cell fate, the next closest cells (P5.p and P7.p) both express the secondary cell fate, and each of the precursor cells located at a distance (P3.p, P4.p and P8.p) express the tertiary cell fate (Fig. 1a). We present data indicating that this stereotypical pattern of cell fates can be generated by sequential signals. We identified genetic mosaic animals in which P5.p and P7.p were defective in the anchor-cell signal-transduction pathway and observed that these cells adopted the secondary cell fate, indicating that anchor-cell signal transduction is not required for the expression of the secondary cell fate. These results suggest that the anchor cell induces P6.p to express the primary cell fate, and that P6.p subsequently induces P5.p and P7.p to express the secondary cell fate.
During vulval development in the Caenorhabditis elegans hermaphrodite, the fates of six vulval precursor cells (VPCs) are influenced by distinct cell signaling events. In one event, a somatic gonadal cell, the anchor cell, induces the three nearest VPCs to adopt vulval cell fates. In another event, lateral signaling between adjacent VPCs specifies one of two different vulval fates, 1 degrees and 2 degrees. Induction of vulval fates by the anchor cell is mediated by a signal transduction pathway involving let-60 Ras, lin-45 Raf, and mpk-1/sur-1 MAP kinase, whereas lateral signaling is mediated by lin-12. We have shown that the mutant phenotype of lin-25, a gene required for VPC fate specification, results from a defect in vulval induction. Genetic epistasis experiments indicate that lin-25 is required in the inductive signaling pathway downstream of let-60 Ras and the Raf/MAP kinase cascade. A decrease in induction also appears to decrease lateral signaling. We have cloned and sequenced the lin-25 gene and shown that it encodes a novel protein that may be a target of the mpk-1/sur-1 MAPK.
The lin-12/Notch proteins appear to act as transmembrane receptors for intercellular signals that specify cell fates during animal development. Recent structure/function studies have shown that the lin-12/Notch intracellular domain alone has the intrinsic signal-transducing activity of the intact protein, and that the role of the extracellular domain is to regulate this intrinsic activity. These studies have also suggested that the different lin-12/Notch proteins in a given organism are interchangeable biochemically and have addressed the role of lin-12/Notch genes in development.
The lin-12 gene of Caenorhabditis elegans is thought to encode a receptor for intercellular signals that specify certain cell fates during development. We describe several alleles of lin-12 that reduce but do not eliminate lin-12 activity (hypomorphic alleles). These alleles cause a novel egg-laying defective (Egl) phenotype in hermaphrodites as well as incompletely penetrant cell fate transformations seen with high penetrance in lin-12 null mutants. Characterization of the Egl phenotype revealed additional roles of lin-12 in the development of the egg-laying system that were not apparent from studying lin-12 null mutants: lin-12 activity is required for proper early vulval morphogenesis as well as for some unknown later aspect of egg-laying system development. Reversion of the Egl phenotype caused by one lin-12 hypomorphic allele was used to identify potential interacting genes as described in the accompanying paper.