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APL-1, a Caenorhabditis elegans protein related to the human beta-amyloid precursor protein, is essential for viability
Abstract
Dominant mutations in the amyloid precursor protein (APP) gene are associated with rare cases of familial Alzheimer's disease; however, the normal functions of APP and related proteins remain unclear. The nematode Caenorhabditis elegans has a single APP-related gene, apl-1, that is expressed in multiple tissues. Loss of apl-1 disrupts several developmental processes, including molting and morphogenesis, and results in larval lethality. The apl-1 lethality can be rescued by neuronal expression of the extracellular domain of APL-1. These data highlight the importance of the extracellular domain of an APP family member and suggest that APL-1 acts noncell-autonomously during development. Overexpression of APL-1 also causes several defects, including a high level of larval lethality. Decreased activity of sel-12, a C. elegans homologue of the human gamma-secretase component presenilin 1, partially rescues the lethality associated with APL-1 overexpression, suggesting that SEL-12 activity regulates APL-1 activity either directly or indirectly.
... 40 This, of course, was 4 years before the completion of its genome sequencing led to the discovery of the C. elegans APP homolog, apl-1, which is expressed in multiple tissue types and is now known to be essential for viability of the organism. 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 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. ...
... 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. 44 ...
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.
... Among the AD-related genes lacking orthologues in C. elegans, β-secretase (BACE1) is worth noting. Although C. elegans has a functional γ-secretase complex [15,16], the absence of β-secretase prevents the cleavage necessary to endogenously produce the β-amyloid peptide in C. elegans. Thus, in the nematode, APL-1 is first cleaved by α-secretase, releasing sAPL-1, and then by the γ-secretase complex [15,16]. ...
... Although C. elegans has a functional γ-secretase complex [15,16], the absence of β-secretase prevents the cleavage necessary to endogenously produce the β-amyloid peptide in C. elegans. Thus, in the nematode, APL-1 is first cleaved by α-secretase, releasing sAPL-1, and then by the γ-secretase complex [15,16]. In addition, the orthologue of APP in C. elegans, apl-1, does not possess the sequence of the β-amyloid peptide, as is the case with the redundant human amyloid β precursor-like proteins 1 and 2 (APLP1 and APLP2). ...
... APL-1 is thus a multifunctional protein involved in molting, reproduction, locomotion and morphogenesis. Moreover, its extracellular domain (sAPL-1) is particularly important, able to act noncell-autonomously and may have essential functions in the nervous system [15]. Loss of apl-1 also independently generated defects in cholinergic neurotransmission at the synaptic level, which could also be rescued by sAPL-1 [83]. ...
Alzheimer’s disease (AD) is the most frequent cause of dementia. After decades of research, we know the importance of the accumulation of protein aggregates such as β-amyloid peptide and phosphorylated tau. We also know that mutations in certain proteins generate early-onset Alzheimer’s disease (EOAD), and many other genes modulate the disease in its sporadic form. However, the precise molecular mechanisms underlying AD pathology are still unclear. Because of ethical limitations, we need to use animal models to investigate these processes. The nematode Caenorhabditis elegans has received considerable attention in the last 25 years, since the first AD models overexpressing Aβ peptide were described. We review here the main results obtained using this model to study AD. We include works studying the basic molecular mechanisms of the disease, as well as those searching for new therapeutic targets. Although this model also has important limitations, the ability of this nematode to generate knock-out or overexpression models of any gene, single or combined, and to carry out toxicity, recovery or survival studies in short timeframes with many individuals and at low cost is difficult to overcome. We can predict that its use as a model for various diseases will certainly continue to increase.
... The Amyloid Precursor Protein (APP) belongs to a family of type I, single pass transmembrane proteins that are highly conserved from mammals to C. elegans, and that have prominent expression in the nervous system (Hornsten et al., 2007;Lorent et al., 1995;Luo et al., 1990;Wiese et al., 2010). Some of the studied family members include APP, APLP1, and APLP2 in mammals (Goldgaber et al., 1987;Kang et al., 1987;Slunt et al., 1994;Tanzi et al., 1987;Wasco et al., 1992Wasco et al., , 1993; APPa, APPb, APLP1, and APLP2 in zebrafish (Liao et al., 2012;Musa et al., 2001); APPL in Drosophila (Luo et al., 1990;Rosen et al., 1989); and APL-1 in C. elegans (Daigle and Li, 1993). ...
... Loss of function studies in mice demonstrated that the APP family is essential and partially redundant: while the single knockouts and App/Aplp1 double knockouts are viable, App/Aplp2 and Aplp1/Aplp2 double knockouts and the triple knockouts die within a few days after birth (Heber et al., 2000;von Koch et al., 1997;Zheng et al., 1995). The single ortholog of App in C. elegans, apl-1, is also an essential gene (Hornsten et al., 2007;Wiese et al., 2010). By contrast, Appl mutants in Drosophila are viable (Luo et al., 1992), and the effect of multiple knockouts in zebrafish is not known. ...
... To assess the physiological role of APP family proteins in mature neurons, we used the GABAergic motor neurons in C. elegans as a model ( Figure 1A). The single C. elegans APP ortholog, apl-1, is known to be expressed in various cell types including neurons, hypodermis, and a number of junction cells (Hornsten et al., 2007;Wiese et al., 2010). We examined apl-1 expression using a Papl-1::mCherry reporter driven by 6.3 kb of the apl-1 promoter, in the juIs76 strain expressing GFP under the control of a GABAergic neuron-specific promoter Punc-25 (Jin et al., 1999). ...
Members of the Amyloid Precursor Protein (APP) family have important functions during neuronal development. However, their physiological functions in the mature nervous system are not fully understood. Here we use the C. elegans GABAergic motor neurons to study the post-developmental function of the APP-like protein APL-1 in vivo. We find that apl-1 has minimum roles in the maintenance of gross neuron morphology and function. However, we show that apl-1 is an inhibitor of axon regeneration, acting on mature neurons to limit regrowth in response to injury. The small GTPase Rab6/RAB-6.2 also inhibits regeneration, and does so in part by maintaining protein levels of APL-1. To inhibit regeneration, APL-1 functions via the E2 domain of its ectodomain; the cytoplasmic tail, transmembrane anchoring, and the E1 domain are not required for this function. Our data defines a novel role for APL-1 in modulating the neuronal response to injury.
... The sequences of Apl-1 genes are highly alike compared to that of humans [48]: E1 and E2 domains share approximately 50% similar sequences with humans, and the transmembrane domain emerges 71% similarity. However, the Apl-1 gene in C.elegans does not produce Aβ sequence [47], allowing this model capable of being tested about the existence and deposition of Aβ sequence on muscle walls when the worm gets paralyzed. ...
... Similarly, Apl-1 shows equivalent outcomes when it's inactivated in C.elegans. During the Larvae stage, C.elegans is observed to be mortal revealed from the inactivation of Apl-1, highlighting the indispensability of APP family in the upgrowth journey of C.elegans, especially molting cycle and motor ability in early stages [48]. Figure 2 [48] shows some morphological defects in C.elegans caused by the abnormality of Apl-1 expression. ...
... During the Larvae stage, C.elegans is observed to be mortal revealed from the inactivation of Apl-1, highlighting the indispensability of APP family in the upgrowth journey of C.elegans, especially molting cycle and motor ability in early stages [48]. Figure 2 [48] shows some morphological defects in C.elegans caused by the abnormality of Apl-1 expression. Fig.2. ...
Alzheimer’s disease (AD) is affecting numerous families and individuals around the world nowadays, as the exact reason is still undetermined. At this stage, developmental treatment displays a particularly significant role in relieving symptoms for the patients. Currently, the two most well-known factors that have impacts on the diagnosis of AD are the plaques and tangles formed from amyloid-beta and tau protein. Modelling for Alzheimer’s disease is essential in understanding targeted aspects of the disease, while Caenorhabditis elegans (C.elegans) was chosen as a pivotal model. C.elegans presents dramatic priorities using orthologs for the study of AD, especially in examining the formation of the deposits and the regulations of specific gene expressions that result in this abnormality. This review discusses the properties, which C.elegans shows on the study of AD, and the achievements that have been approached using this model, as well as what other models are being tested by scientists. Properties of other models, which can overwhelm C.elegans, as well as the expectations for future modelling systems on AD are examined as well.
... C. elegans also shows similar effects of air pollution in the responsiveness of its homologous amyloid processing genes to nPM. Several amyloid-related genes are associated with C. elegans development; for example, inactivation of apl-1/APP results in penetrant lethality during the L1 to L2 transition due to molting defects (44). On the other hand, overexpression of apl-1/APP results in penetrant L1 lethality, shortened body length and morphological, locomotive, and reproductive effects (44,45). ...
... Several amyloid-related genes are associated with C. elegans development; for example, inactivation of apl-1/APP results in penetrant lethality during the L1 to L2 transition due to molting defects (44). On the other hand, overexpression of apl-1/APP results in penetrant L1 lethality, shortened body length and morphological, locomotive, and reproductive effects (44,45). sel-12/PSEN is one of the genes regulating apl-1/APP cleavage and trafficking (44). ...
... On the other hand, overexpression of apl-1/APP results in penetrant L1 lethality, shortened body length and morphological, locomotive, and reproductive effects (44,45). sel-12/PSEN is one of the genes regulating apl-1/APP cleavage and trafficking (44). Examining the interaction of different life stages on nPM (50 g/ml) mediated hormesis effects (n=3 52-101/group) and the role of skn-1 response in this process until 90% survival in wild type non-exposed animals (n=2-3 47-53/group) (Summary statistics in Table S2 Table S3 for summary statistics of multivariate models. ...
Air pollution is a heterogeneous environmental toxicant that impacts humans throughout their life. We introduce Caenorhabditis elegans as a valuable air pollution model with its short lifespan, medium-throughput capabilities, and highly conserved biological pathways that impact healthspan. We exposed developmental and adult life stages of C. elegans to airborne nano-sized particulate matter (nPM) produced by traffic emissions and measured biological and molecular endpoints that changed in response. Acute nPM did not cause lethality in C. elegans, but short-term exposure during larval stage 1 caused delayed development. Gene expression responses to nPM exposure overlapped with responses of mouse and cell culture models of nPM exposure in previous studies. We showed further that the skn-1/Nrf2 antioxidant response has a role in the development and hormetic effects of nPM. This study introduces the worm as a new resource and complementary model for mouse and cultured cell systems to study air pollution toxicity across the lifespan.
... Similar to human APP, C. elegans APL-1 is a single transmembrane-spanning protein with a large extracellular and a small intracellular domain, both of which share sequence homology to human APP (Daigle & Li, 1993). Analogous to the postnatal lethality of APP family-knockout mice, loss of apl-1 in C. elegans results in a completely penetrant larval lethality that is rescued by the reintroduction of either full-length APL-1 or only the extracellular domain of APL-1 (Hornsten et al., 2007). This finding was later extended to mammals, whereby knockin of sAPPa rescued the postnatal lethality of the APP-APLP2double-knockout mice (Weyer et al., 2011). ...
... This finding was later extended to mammals, whereby knockin of sAPPa rescued the postnatal lethality of the APP-APLP2double-knockout mice (Weyer et al., 2011). Furthermore, overexpression of APL-1 in C. elegans also causes an early developmental lethality, but the lethality shows an incomplete penetrance that is correlated with higher levels of APL-1 (Hornsten et al., 2007). Similarly, overexpression of human or mouse APP in mice causes early developmental lethality in the absence of amyloid plaques (Hsiao et al., 1995). ...
... Ectopic expression of APL-1 during adulthood increases lifespan High levels of APL-1 cause larval lethality (Hornsten et al., 2007). To bypass the developmental effects of APL-1 and to determine the role of APL-1 during aging, we used transgenic animals (ynIs14) that have the hsp-16.2 ...
Alzheimer's disease (AD) is an age-associated disease. Mutations in the amyloid precursor protein (APP) may be causative or protective of AD. The presence of two functionally redundant APP-like genes (APLP1/2) has made it difficult to unravel the biological function of APP during aging. The nematode Caenorhabditis elegans contains a single APP family member, apl-1. Here, we assessed the function of APL-1 on C. elegans' lifespan and found tissue-specific effects on lifespan by overex-pression of APL-1. Overexpression of APL-1 in neurons causes lifespan reduction, whereas overexpression of APL-1 in the hypodermis causes lifespan extension by repressing the function of the heterochronic transcription factor LIN-14 to preserve youthfulness. APL-1 lifespan extension also requires signaling through the FOXO transcription factor DAF-16, heat-shock factor HSF-1, and vitamin D-like nuclear hormone receptor DAF-12. We propose that reinforcing APL-1 expression in the hypodermis preserves the regulation of heterochronic lin-14 gene network to improve maintenance of somatic tissues via DAF-16/FOXO and HSF-1 to promote healthy aging. Our work reveals a mechanistic link of how a conserved APP-related protein modulates aging.
... Two APP KO mice have been extensively characterised, a homozygous APP null mutant (Zheng et al., 1995) and an APP deficient mouse (APPD) in which only 5% of normal APP is expressed (the majority of APP transcripts represent a shorter form due to a deletion of APP exon 2) (Muller et al., 1994). Other in vivo KO models include Drosophila lacking APPL (Luo et al., 1992;Torroja et al., 1999;Gunawardena and Goldstein, 2001) and Caenorhabditis elegans lacking apl-1 (Hornsten et al., 2007), both orthologues of human APP. ...
... Amyloid precursor protein KO mice and flies are viable and fertile (Luo et al., 1992;Muller et al., 1994;Zheng et al., 1995); however, lack of APPL in Drosophila leads to shorter lifespan (Wentzell et al., 2012) while loss of apl-1 in C. elegans results in larval lethality (Hornsten et al., 2007). Nevertheless, in mice, APP-family members have redundant functions and may play a compensatory role (see Section Amyloid Precursor Protein and APLP Redundancy). ...
Amyloid precursor protein (APP) and its cleavage fragment Amyloid-β (Aβ) have fundamental roles in Alzheimer’s disease (AD). Genetic alterations that either increase the overall dosage of APP or alter its processing to favour the generation of longer, more aggregation prone Aβ species, are directly causative of the disease. People living with one copy of APP are asymptomatic and reducing APP has been shown to lower the relative production of aggregation-prone Aβ species in vitro. For these reasons, reducing APP expression is an attractive approach for AD treatment and prevention. In this review, we will describe the structure and the known functions of APP and go on to discuss the biological consequences of APP knockdown and knockout in model systems. We highlight progress in therapeutic strategies to reverse AD pathology via reducing APP expression. We conclude that new technologies that reduce the dosage of APP expression may allow disease modification and slow clinical progression, delaying or even preventing onset.
... Several of the worm genes that we identified have established roles in the nervous system, including apl-1 (APP) and hlh-16 (OLIG1 and OLIG2) (Bertrand, Bisso, Poole, & Hobert, 2011;Ewald et al., 2012;Hornsten et al., 2007) . We also identified several genes associated with defects in locomotion that are less clearly linked to the nervous system in worm. ...
... It is made available under The copyright holder for this preprint (which was not this version posted May 11, 2017. an essential gene that functions within the nervous system (Hornsten et al., 2007). Its closest human HSA21 ortholog, APP (amyloid precursor protein) is a key gene associated with the development of the neurodegenerative disorder Alzheimer's disease, including in people with Down syndrome and in a DS mouse model (Salehi et al., 2006). ...
Individuals with Down syndrome have neurological and muscle impairments due to an additional copy of the human 21 st chromosome (HSA21). Only a few of ~200 HSA21 genes encoding protein have been linked to specific Down syndrome phenotypes, while the remainder are understudied. To identify poorly characterized HSA21 genes required for nervous system function, we studied behavioral phenotypes caused by loss-of-function mutations in conserved HSA21 orthologs in the nematode Caenorhabditis elegans . We identified ten HSA21 orthologs that are required for neuromuscular behaviors: cle-1 ( COL18A1 ), cysl-2 ( CBS ), dnsn-1 ( DONSON ), eva-1 ( EVA1C ), mtq-2 ( N6ATM1 ), ncam-1 ( NCAM2 ), pad-2 ( POFUT2 ), pdxk-1 ( PDXK ), rnt-1 ( RUNX1 ), and unc-26 ( SYNJ1 ). We also found that three of these genes are required for normal release of the neurotransmitter acetylcholine. This includes a known synaptic gene unc-26 ( SYNJ1 ), as well as uncharacterized genes pdxk-1 ( PDXK ) and mtq-2 ( N6ATM1 ). As the first systematic functional analysis of HSA21 orthologs, this study may serve as a platform to understand genes that underlie phenotypes associated with Down syndrome.
ARTICLE SUMMARY
Down syndrome causes neurological and muscle dysfunction due to an extra 21 st chromosome. This chromosome has over 200 genes, most of which are understudied. To address this, we studied whether reducing function of these gene equivalents in the worm C. elegans caused neuronal or muscle defects. We identified ten genes conserved between human and worm that mediate function of behaviors. Among these, we show the uncharacterized genes mtq-2 and pdxk-1 are important for synaptic transmission and are exclusively expressed in nervous system. Our analysis may reveal functions of poorly studied genes that affect nervous system function in Down syndrome.
... Like the mammalian protein, the C. elegans APP protein (APL-1) is expressed in a variety of cells, including neurons, muscles, and hypodermal cells (Hornsten et al., 2007). Loss of APL-1 results in lethality during development, which can be rescued by expression of the soluble N-terminal fragment alone, suggesting that the N-terminus is sufficient for the vital function of APL-1 (Hornsten et al., 2007). ...
... Like the mammalian protein, the C. elegans APP protein (APL-1) is expressed in a variety of cells, including neurons, muscles, and hypodermal cells (Hornsten et al., 2007). Loss of APL-1 results in lethality during development, which can be rescued by expression of the soluble N-terminal fragment alone, suggesting that the N-terminus is sufficient for the vital function of APL-1 (Hornsten et al., 2007). In contrast, expression of the Drosophila APP protein ...
Alzheimer’s disease is a major and increasing burden on families, communities, and national health budgets. Despite intensive and extended research there is still widespread debate about its cause(s) and no effective treatments exist. Familial (inherited, mainly early onset) and sporadic (mainly late onset) forms of the disease exist and it is uncertain to what extent they are related. Transgenic mouse models have dominated the investigation of this disease but their validity can be questioned. Numerous alternative models exist that can provide valuable information on the molecular and cellular basis of Alzheimer’s disease. In this chapter we review the various invertebrate, nonmammalian vertebrate, and mammalian models and how these have been used to investigate this disease. We examine the strengths and weaknesses of these various model systems. Of course, animal models never completely reflect the true nature of a human disease but progress in understanding and finding preventative and ameliorative treatments for Alzheimer’s disease is hindered by the lack of a convincing hypothesis for the cause of this complex condition.
... The brood size assay showed that there was no significant increase in brood size for any strains, microinjected with LRP1 or AQP4, or both (Figure 4). This was unexpected since the expression of beta amyloid is known to decrease the brood size, and therefore with the addition of LRP1 and AQP4, the brood size should have increased (14). Brood size may have increased growth after a few days and could be tested in the future. ...
Alzheimer’s is ranked as the 6th leading cause of death in the United States, and mainly presented as neurodegeneration. In order to begin to understand its physiology, the specific role of proteins in neurodegeneration, LRP1 and AQP4, need to be studied. This study tested the effect of using the CRISPR-Cas9 system to overexpress the LRP1 and AQP4 proteins, associated with transport of materials and waste across the cell membrane, in Caenorhabditis elegans to assess effects on neurodegeneration, such as chemosensation, size, and average speed phenotypes. I hypothesized that combinatorial overexpression of AQP4 and LRP1 would have the greatest effect on reducing neurodegeneration. I tested chemosensory behavior using a chemotaxis test, revealing a decrease in neurodegeneration when both LRP1 and AQP4 were overexpressed. The size of the C. elegans did not change, but the speed increased in the strain expressing amyloid beta in the muscle, suggesting that a decrease in amyloid beta allowed muscles more room to contract. These results support our hypothesis and show that the overexpression of LRP1 and AQP4 proteins decrease neurodegeneration and allow C. elegans to preserve their olfactory retention. This study will help demonstrate the role of LRP1 and AQP4 in Alzheimer’s and determine whether they benefit the system once they are overexpressed.
... Aβ is a small peptide produced by the sequential enzymatic processing of APP. The C. elegans genome encodes for a single APP protein (APL-1), which is an ortholog of human APLP1-2 (amyloid beta precursor-like) proteins [50]. Single-copy pan-neuronal expression of human APP, in addition to the endogenous ALP-1 of the nematode, results in neurodegeneration and neurobehavioral dysfunction [51]. ...
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.
... Important clues for the role of APP in development comes from organisms that have only a single APP family member. In C. elegans, APL-1 knockout is lethal due to a molting defect (Hornsten et al., 2007;Wiese, Antebi and Zheng, 2010). APPL knockout Drosophila is viable but have minor defects including conditional learning defects (Luo, Tully and White, 1992), defects in maintenance of synaptic boutons at the neuromuscular junction (NMJ) (Torroja et al., 1999), more vulnerability to brain injury and defect in the endo-lysosomal pathway . ...
The neocortex is the highly elaborate part of the human brain which is responsible for complex cognitive behavior. During embryogenesis, human cortical neural progenitor cells (NPCs) initially generate neurons at a particularly slow rate while preserving their progenitor state for a relatively long time, in part contributing to increased human cortical size. The Amyloid Precursor Protein (APP), which is responsible for genetic forms of Alzheimer’s disease, is highly expressed in early embryonic days in human telencephalic neurospheres, along with differentiation, migration and maturation of cortical neurons. However, the role of APP in human context development is completely unexplored. The general introduction of this thesis summarizes previous findings on cortical neurogenesis and the implication of APP in various aspect of neurobiology with a focus on human cortex development. We hypothesized APP may have a role in the development of human cortex and this project outlines a strategy to unveil the physiological functions of human APP during cortical development. We used human induced pluripotent stem cells as a model. We produced APP knock-out iPSC in two different genetic backgrounds and studied cortical neurogenesis in 2D and 3D. We found that loss of APP causes premature differentiation specifically in human cortical progenitors, but the relatively slow timing of neuronal maturation appears preserved. In contrast, we found no effect on mouse cortical progenitors and the rate of human spinal motor neuron generation. Mechanistically, loss of APP cell-autonomously triggers conversion of cortical NPCs to a neurogenic state through repression of the canonical Wnt pathway and upregulation and activation of AP1 (Jun and Fos), the well-known stress response genes. AP1 activates downstream proneural proteins, like Neurogenin2 and B-cell lymphoma 6 (Bcl6) thus inducing neural progenitors to exit the cell cycle and prematurely differentiate. These phenotypes can be fully rescued either by restoring APP, or by adding exogenous Wnt3a to restore NPC proliferation and inhibiting AP1 activity to inhibit premature differentiation. In summary, this project reveals a human cortex specific role for APP in regulating neurogenesis and identifies the regulation of cellular stress as a potential intrinsic mechanism for prolonged maintenance of human cortical progenitor fate.
... The major component of these plaques, β-amyloid peptide (Aβ), is derived from the amyloid precursor protein (APP) through the sequential action of βand γ-secretase [24]. Drosophila melanogaster and C. elegans do not have the APP gene, but both express the amyloid precursor protein gene APL-1 [89], which is controlled by let-7 and its targets. For instance, let-7 transcriptionally regulates APL-1 through Hbl1, Lin-41, and Lin-42 and is critical for the development of AD [90]. ...
The abnormal regulation and expression of microRNA (miRNA) are closely related to the aging process and the occurrence and development of aging-related diseases. Lethal-7 (let-7) was discovered in Caenorhabditis elegans (C. elegans) and plays an important role in development by regulating cell fate regulators. Accumulating evidence has shown that let-7 is elevated in aging tissues and participates in multiple pathways that regulate the aging process, including affecting tissue stem cell function, body metabolism, and various aging-related diseases (ARDs). Moreover, recent studies have found that let-7 plays an important role in the senescence of B cells, suggesting that let-7 may also participate in the aging process by regulating immune function. Therefore, these studies show the diversity and complexity of let-7 expression and regulatory functions during aging. In this review, we provide a detailed overview of let-7 expression regulation as well as its role in different tissue aging and aging-related diseases, which may provide new ideas for enriching the complex expression regulation mechanism and pathobiological function of let-7 in aging and related diseases and ultimately provide help for the development of new therapeutic strategies.
... The catalytic subunits of γ-secretase, PSEN1 and PSEN2, were aligned to C. elegans presenilin orthologs HOP-1 and SEL-12, respectively. HOP-1 and SEL-12 are parts of the γ-secretase complex, just like their human counterparts, with evidence suggesting that SEL-12, directly or indirectly, regulates the activity of APL-1 42 . Overall, most of the APP processing machinery, and its corresponding orthologs, arose as important common elements during this work and were determined to indeed be conserved in the model organism. ...
Alzheimer disease (AD) is a neurodegenerative disorder with an –as of yet– unclear etiology and pathogenesis. Research to unveil disease processes underlying AD often relies on the use of neurodegenerative disease model organisms, such as Caenorhabditis elegans . This study sought to identify biological pathways implicated in AD that are conserved in Homo sapiens and C. elegans . Protein–protein interaction networks were assembled for amyloid precursor protein (APP) and Tau in H. sapiens —two proteins whose aggregation is a hallmark in AD—and their orthologs APL-1 and PTL-1 for C. elegans . Global network alignment was used to compare these networks and determine similar, likely conserved, network regions. This comparison revealed that two prominent pathways, the APP-processing and the Tau-phosphorylation pathways, are highly conserved in both organisms. While the majority of interactions between proteins in those pathways are known to be associated with AD in human, they remain unexamined in C. elegans , signifying the need for their further investigation. In this work, we have highlighted conserved interactions related to AD in humans and have identified specific proteins that can act as targets for experimental studies in C. elegans , aiming to uncover the underlying mechanisms of AD.
... Nevertheless, single-copy insertion and expression of human APP results in neurodegeneration and neurobehavioral dysfunction in C. elegans (Yi et al., 2017). Likewise, although apl-1 is essential for viability, overexpression of apl-1 yields pleiotropic phenotypes that include neuronal deficits (Hornsten et al., 2007;Ewald et al., 2012). Owing to spatial constraints, the AD section of this article will focus on Aβ modeling, while the ALS section will describe tau expression in C. elegans. ...
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.
... However, follow-up studies on Notch signalling's role in AD pathology associated neurodegeneration should be done before definite conclusions can be made. Hornsten et al. (2007) made an interesting observation bringing together sel-12 and apl-1. When sel-12 activity was reduced in transgenic worms with APL-1 over-expression, the lethality phenotype observed in apl-1 GOF mutants was partially rescuedsuggesting that sel-12 may regulate APL-1 cleavage and/or trafficking. ...
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.
... Therefore, impairment of neurogenesis is quite related to the disease progression itself. Beta amyloid precursor protein (βAPP) the precursor of amyloid beta (Aβ) is important for neuron generation, differentiation and neural migration (142,143) as the embryonic expression of βAPP peaks during neuronal differentiation and neurite outgrowth period (144,145). The studies performed on transgenic animals expressing mutant βAPP demonstrated decreased neurogenesis in adult brains (146)(147)(148)(149). Noteworthy is that melatonin levels are decreased in blood and cerebrospinal fluid (CSF) of AD patients and this reduction runs parallel with the progression of AD pathogenesis (150). ...
The revelation of adult brain exhibiting neurogenesis has established that the brain possesses great plasticity and that neurons could be spawned in the neurogenic zones where hippocampal adult neurogenesis attributes to learning and memory processes. With strong implications in brain functional homeostasis, aging and cognition, various aspects of adult neurogenesis reveal exuberant mechanistic associations thereby further aiding in facilitating the therapeutic approaches regarding the development of neurodegenerative processes in Alzheimer’s Disease (AD). Impaired neurogenesis has been significantly evident in AD with compromised hippocampal function and cognitive deficits. Melatonin the pineal indolamine augments neurogenesis and has been linked to AD development as its levels are compromised with disease progression. Here, in this review, we discuss and appraise the mechanisms via which melatonin regulates neurogenesis in pathophysiological conditions which would unravel the molecular basis in such conditions and its role in endogenous brain repair. Also, its components as key regulators of neural stem and progenitor cell proliferation and differentiation in the embryonic and adult brain would aid in accentuating the therapeutic implications of this indoleamine in line of prevention and treatment of AD.
... small GTPase UNM-108 leads to retention of APL-1 in the cell body (Wiese et al., 2010). Overexpression of apl-1 resulted in interference of motor neuron functions including reduced swimming and crawling rates compared to wildtype as well as defects in brood size, and viability which were shown to be correlated with the level of APL-1 (Hornsten et al., 2007). ...
... Evidence from APPL knockout Drosophila has found to be viable but with slight behavioral defects (conditional learning defects) [76] and defects in maintaining the integrity of synaptic boutons at the neuromuscular junctions [77], and was susceptible to brain injury [78]. In another study, APL-1 knockout condition in Caenorhabditis elegans has been found to be lethal due to molting defect [79,80]. APP has a crucial role to play in neuronal migration during early embryogenesis. ...
Amyloid precursor protein (APP) is a transmembrane protein expressed largely within the central nervous system. Upon cleavage, it does not produce the toxic amyloid peptide (Aβ) only, which is involved in neurodegenerative progressions but via a non-amyloidogenic pathway it is metabolized to produce a soluble fragment (sAPPα) through α-secretase. While a lot of studies are focusing on the role played by APP in the pathogenesis of Alzheimer’s disease, sAPPα is reported to have numerous neuroprotective effects and it is being suggested as a candidate with possible therapeutic potential against Alzheimer’s disease. However, the mechanisms through which sAPPα precisely works remain elusive. We have presented a comprehensive review of how sAPPα is regulating the neuroprotective effects in different biological models. Moreover, we have focused on the role of sAPPα during different developmental stages of the brain, neurogenic microenvironment in the brain and how this metabolite of APP is regulating the neurogenesis which is regarded as a compelling approach to ameliorate the impaired learning and memory deficits in dementia and diseases like Alzheimer’s disease. sAPPα exerts beneficial physiological, biochemical and behavioral effects mitigating the detrimental effects of neurotoxic compounds. It has shown to increase the proliferation rate of numerous cell types and promised the synaptogenesis, neurite outgrowth, cell survival and cell adhesion. Taken together, we believe that further studies are warranted to investigate the exact mechanism of action so that sAPPα could be developed as a novel therapeutic target against neuronal deficits.
... spe-4 is only expressed in the male germ line, while hop-1 and sel-12 are widely expressed and sel-12 shows the higher sequence homology to human presenilins. In the same way as presenilins control APP processing, SEL-12 appears to regulate APL-1 cleavage, because apl-1 overexpression produces 70% lethality, and this is rescued in sel-12 mutants [16]. Mutations in sel-12 result in mitochondrial metabolic defects that promote neurodegeneration in the worm as a result of deregulation of mitochondrial Ca 2+ homeostasis [15,17,18] (see below Section 4.1.). ...
Ca2+ is a ubiquitous second messenger that plays an essential role in physiological processes such as muscle contraction, neuronal secretion, and cell proliferation or differentiation. There is ample evidence that the dysregulation of Ca2+ signaling is one of the key events in the development of neurodegenerative processes, an idea called the “calcium hypothesis” of neurodegeneration. Caenorhabditis elegans (C. elegans) is a very good model for the study of aging and neurodegeneration. In fact, many of the signaling pathways involved in longevity were first discovered in this nematode, and many models of neurodegenerative diseases have also been developed therein, either through mutations in the worm genome or by expressing human proteins involved in neurodegeneration (β-amyloid, α-synuclein, polyglutamine, or others) in defined worm tissues. The worm is completely transparent throughout its whole life, which makes it possible to carry out Ca2+ dynamics studies in vivo at any time, by expressing Ca2+ fluorescent probes in defined worm tissues, and even in specific organelles such as mitochondria. This review will summarize the evidence obtained using this model organism to understand the role of Ca2+ signaling in aging and neurodegeneration.
... Moreover, neurotransmitters and other components of the nervous system are conserved between the nematode and mammals (Driscoll and Gerstbrein, 2003). Despite the fact that C. elegans misses adequate homologues for human APP and β-secretase genes, mechanisms of AD can still be studied in the nematode: Several transgenic strains expressing human Aβ peptides in muscle cells or neurons have been engineered (Link, 1995;Hornsten et al., 2007). This article summarizes common C. elegans models for Aβ toxicity and how they have been used to study the molecular pathomechanism and possible pharmaceuticals Duan & Sesti 2013 Aβ: amyloid beta peptide; aex-3: encoding a guanine nucleotide exchange factor for the rab-3 GTPase which is required for intracellular vesicle trafficking and synaptic vesicle release; eat-4: encodes an ortholog of the mammalian BNPI vesicular glutamate transporter; gcy-2: guanylyl cyclase, sensory neuron receptor; flp-6: expressed in ASI neurons; mtl-2: metallothionein; myo-2: muscle myosin class II heavy chain C (MHC), expressed in pharynx muscles; myo-3: MHC A, expressed exclusively in the body wall muscle; rol-6: encodes a cuticle collagen related to human collagen alpha 1 (III) chain precursor; snb-1: synaptobrevin, codes for a synaptic vesicle protein (orthologous to human vesicle-associated membrane protein 1); ttx-3: LIM/homeobox protein, AIA and AIY interneurons and the NSM neurons; required for functions of the interneuron AIY, unc-54: MHC B, exclusively in the body wall muscle; unc-119: pan-neuronally expressed, function not known. ...
Neurodegenerative diseases are a group of chronic, progressive disorders characterized by neuronal loss and symptoms related to the affected brain or medulla areas. They affect millions of people worldwide and because their mechanisms are not totally understood, treatments are still experimental. In order to further study these diseases and how to treat them, Caenorhabditis elegans has arisen as an alternative/complementary animal model, well recognized by the scientific community, notably because of its neurophysiology, very much alike from mammals, but elementary. This nematode has 302 neurons, including dopaminergic, serotoninergic, cholinergic and GABAergic. The formation, trafficking and release of neurotransmitters from the synaptic vesicles are highly conserved processes in relation to mammals. In addition, strains that express human mutated proteins that aggregate as huntingtin, amiloyd β and α-synuclein have been successfully generated. Because of these attractive advantages, many researchers have adopted this worm to study and understand how neurodegeneration processess start and progress. In this chapter we will discuss the neurodegenerative diseases models that have been developed in C. elegans and also regarding neurotoxicity induced by toxicants such as metals, pesticides, and nanoparticles, which have been associated to gene x environment interactions in neurodegenerative disorders.
... MEX-3/APL-1-mediated neuronal patterning and MEX-3/CDC-14-mediated cell fate determination in the MEX-3-mediated gene cascade. Because MEX-3 is specifically expressed in AB cells at the four-cell stage, spatiotemporal-regulated synaptic formation defects in hbl-1 mutants and apl-1-dependent embryonic neuronal patterning may be elucidated by identifying MEX-3/APL-1-mediated gene cascades (Fig 3E) [54][55][56]. In parallel, when we focused on the MEX-3/CDC-14-mediated gene cascade (Fig 3E), CDC-14B, a zebrafish homolog of CDC-14, was shown to be involved in formation of the cilium in sensory neurons [55]. ...
Obtaining a comprehensive understanding of the gene regulatory networks, or gene cascades, involved in cell fate determination and cell lineage segregation in Caenorhabditis elegans is a long-standing challenge. Although RNA-sequencing (RNA-Seq) is a promising technique to resolve these questions, the bioinformatics tools to identify associated gene cascades from RNA-Seq data remain inadequate. To overcome these limitations, we developed Gene Cascade Finder (GCF) as a novel tool for building gene cascades by comparison of mutant and wild-type RNA-Seq data along with integrated information of protein-protein interactions, expression timing, and domains. Application of GCF to RNA-Seq data confirmed that SPN-4 and MEX-3 regulate the canonical Wnt pathway during embryonic development. Moreover, lin-35, hsp-3, and gpa-12 were found to be involved in MEX-1-dependent neurogenesis, and MEX-3 was found to control the gene cascade promoting neurogenesis through lin-35 and apl-1. Thus, GCF could be a useful tool for building gene cascades from RNA-Seq data.
... Binding interface of Amyloid Precursor Protein (APP) and Contactin3 (CNTN3) Invertebrate genomes also code for APP and CNTN family members. Conveniently, the two best studied invertebrate model organisms, C. elegans and D. melanogaster, each have only a single APP homologue (APL-1 in C. elegans [32] and APPL in D. melanogaster [33]) and a single CNTN homologue (RIG-6 in C. elegans [34] and CONT in D. melanogaster [35]). At present, there are no reports of APP-CNTN interactions in C. elegans or D. melanogaster. ...
The Amyloid Precursor Protein (APP) and Contactin (CNTN) families of cell-surface proteins have been intensively studied in the context of neural development and neuropsychiatric diseases. Earlier studies demonstrated both genetic and biochemical interactions between the extracellular domains of APP and CNTN3, but their precise binding interfaces were not defined. In the present study, we have used binding assays between APP-alkaline phosphatase (AP) fusion proteins and CNTN-Fc fusion proteins, together with alanine substitution mutagenesis, to show that: (i) the second Fibronectin domain (Fn(2)) in CNTN3 mediates APP binding; (ii) the copper binding domain (CuBD) in APP mediates CNTN3 binding; and (iii) the most important amino acids for APP-CNTN3 binding reside on one face of CNTN3-Fn(2) and on one face of APP-CuBD. These experiments define the regions of direct contact that mediate the binding interaction between APP and CNTN3.
... Moreover, neurotransmitters and other components of the nervous system are conserved between the nematode and mammals (Driscoll and Gerstbrein, 2003). Despite the fact that C. elegans misses adequate homologues for human APP and β-secretase genes, mechanisms of AD can still be studied in the nematode: Several transgenic strains expressing human Aβ peptides in muscle cells or neurons have been engineered (Link, 1995;Hornsten et al., 2007). This article summarizes common C. elegans models for Aβ toxicity and how they have been used to study the molecular pathomechanism and possible pharmaceuticals Duan & Sesti 2013 Aβ: amyloid beta peptide; aex-3: encoding a guanine nucleotide exchange factor for the rab-3 GTPase which is required for intracellular vesicle trafficking and synaptic vesicle release; eat-4: encodes an ortholog of the mammalian BNPI vesicular glutamate transporter; gcy-2: guanylyl cyclase, sensory neuron receptor; flp-6: expressed in ASI neurons; mtl-2: metallothionein; myo-2: muscle myosin class II heavy chain C (MHC), expressed in pharynx muscles; myo-3: MHC A, expressed exclusively in the body wall muscle; rol-6: encodes a cuticle collagen related to human collagen alpha 1 (III) chain precursor; snb-1: synaptobrevin, codes for a synaptic vesicle protein (orthologous to human vesicle-associated membrane protein 1); ttx-3: LIM/homeobox protein, AIA and AIY interneurons and the NSM neurons; required for functions of the interneuron AIY, unc-54: MHC B, exclusively in the body wall muscle; unc-119: pan-neuronally expressed, function not known. ...
Frailty is generally defined as the increased susceptibility of individuals of the same chronological age to adverse outcomes. Frailty has been recently considered as an independent identifiable clinical outcome and is characterized by, but not restricted to, weight loss, physical exhaustion, reduced walking speed, weakness, age-related diseases and cognitive impairment. In humans, frailty arises from genetic and environmental interactions, leading to multisystemic physiological decline. However, the molecular basis of this important age-related condition is far from understood. The nematode Caenorhabditis elegans is commonly used as a model of aging due to its short lifespan and well characterized genetics. In addition, environmental manipulations such as different dietary regimens, exposure to drugs or toxins, and temperature variation affect the worm’s aging process. Thus, a broad genetic and metabolic overview of how aging can be manipulated is provided by this model organism. Considering the clinical relevance of frailty, in the last years aging research has shifted focus from interventions that modulate lifespan to ones that promote healthspan, i.e. altering frailty-free living time. To model that in the nematode, physiological parameters are measured across lifespan at different experimental settings. These parameters include locomotion, feeding behavior, neuromuscular communication, intestinal barrier integrity and stress resistance. This chapter will describe the main techniques used to score frailty in C. elegans and provide an update of the new discoveries regarding the molecular mechanisms of frailty using this organism as a tool.
... 19,20 In C. elegans, apl-1 has been shown to be involved in neuronal plasticity, pharyngeal pumping, and molting. [21][22][23] Notably, Niwa et al. showed that apl-1 suppresses the let-7 retarded phenotypes and genetically interacts with hbl -1, lin-41, and lin-42. 18 In this study, we aimed to further elucidate the role of apl-1 in developmental timing in order to both gain insights into the mechanisms of the heterochronic pathway and potentially elucidate the function of an Alzheimer's disease-associated gene. ...
The heterochronic pathway in C. elegans controls the relative timing of cell fate decisions during post-embryonic development. It includes a network of microRNAs (miRNAs), such as let-7, and protein-coding genes, such as the stemness factors, LIN-28 and LIN-41. Here we identified the acn-1 gene, a homologue of mammalian angiotensin-converting enzyme (ACE), as a new suppressor of the stem cell developmental defects of let-7 mutants. Since acn-1 null mutants die during early larval development, we used RNAi to characterize the role of acn-1 in C. elegans seam cell development, and determined its interaction with heterochronic factors, including let-7 and its downstream interactors – lin-41, hbl-1, and apl-1. We demonstrate that although RNAi knockdown of acn-1 is insufficient to cause heterochronic defects on its own, loss of acn-1 suppresses the retarded phenotypes of let-7 mutants and enhances the precocious phenotypes of hbl-1, though not lin-41, mutants. Conversely, the pattern of acn-1 expression, which oscillates during larval development, is disrupted by lin-41 mutants but not by hbl-1 mutants. Finally, we show that acn-1(RNAi) enhances the let-7-suppressing phenotypes caused by loss of apl-1, a homologue of the Alzheimer's disease-causing amyloid precursor protein (APP), while significantly disrupting the expression of apl-1 during the L4 larval stage. In conclusion, acn-1 interacts with heterochronic genes and appears to function downstream of let-7 and its target genes, including lin-41 and apl-1.
... Indeed, its deletion or reduction is associated with impaired neurite outgrowth, decreased neuronal viability, and reduced synaptic Diagnostics 2017, 7, 42 4 of 21 activity [28,29]. The growth promoting, synaptotrophic and neuroprotective functions may be exerted, in part, both by the full-length APP protein and by its α-secretase cleaved soluble fragment APPsα [30]. In contrast, the slightly shorter β-secretase cleavage product (APPsβ) is considerably less active and may even be neurotoxic [31]. ...
Alzheimer's disease is the most common cause of dementia. Over the last three decades, research has advanced dramatically and provided a detailed understanding of the molecular events underlying the pathogenesis of Alzheimer's disease. In parallel, assays for the detection of biomarkers that reflect the typical Alzheimer's disease-associated pathology have been developed and validated in myriads of clinical studies. Such biomarkers complement clinical diagnosis and improve diagnostic accuracy. The use of biomarkers will become even more important with the advent of disease-modifying therapies. Such therapies will likely be most beneficial when administered early in the disease course. Here, we summarise the development of the core Alzheimer's disease cerebrospinal fluid biomarkers: amyloid-β and tau. We provide an overview of their role in cellular physiology and Alzheimer's disease pathology, and embed their development as cerebrospinal fluid biomarkers into the historical context of Alzheimer's disease research. Finally, we summarise recommendations for their use in clinical practice, and outline perspectives for novel cerebrospinal fluid candidate biomarkers.
... APP is highly conserved across different phyla including mammals, insects and nematodes, suggesting that the protein has advantageous effects on survival and reproduction of animals (Müller and Zheng, 2012;van der Kant and Goldstein, 2015). Indeed, in the nematode C. elegans knock-out of APP-like protein (APL-1) is lethal (Hornsten et al., 2007). Drosophila lacking the APP ortholog APPL exhibit severe memory deficits (Bourdet et al., 2015). ...
Despite its key role in the molecular pathology of Alzheimer’s disease (AD), the physiological function of amyloid precursor protein (APP) is unknown. Increasing evidence, however, points towards a neuroprotective role of this membrane protein in situations of metabolic stress. A key observation is the up-regulation of APP following acute (stroke, cardiac arrest) or chronic (cerebrovascular disease) hypoxic-ischemic conditions. While this mechanism may increase the risk or severity of AD, APP by itself or its soluble extracellular fragment APPsα can promote neuronal survival. Indeed, different animal models of acute hypoxia-ischemia, traumatic brain injury (TBI) and excitotoxicity have revealed protective effects of APP or APPsα. The underlying mechanisms involve APP-mediated regulation of calcium homeostasis via NMDA receptors (NMDAR), voltage-gated calcium channels (VGCC) or internal calcium stores. In addition, APP affects the expression of survival- or apoptosis-related genes as well as neurotrophic factors. In this review, we summarize the current understanding of the neuroprotective role of APP and APPsα and possible implications for future research and new therapeutic strategies.
... Based on early work suggesting that APP might regulate both cell adhesion and excitoprotective responses (Mattson et al., 1993; Schubert and Behl, 1993), a variety of in vitro and in vivo assays demonstrated that both full-length APP and its sAPPα ectodomain fragments (produced by α-secretase cleavage) could have potent neuroprotective activity under different conditions (reviewed in Kögel et al., 2012; Nhan et al., 2015). For example, deletion of the sole APP ortholog in nematode (APL-1) caused larval lethality that could be rescued by expressing extracellular domain fragments equivalent to sAPPα (Hornsten et al., 2007; Ewald et al., 2016), while overexpressing sAPPα rescued some behavioral deficits in mice lacking members of the APP family (Ring et al., 2007; Weyer et al., 2011). From these and other experiments emerged a complex scenario whereby both APP and sAPPα might independently confer beneficial responses under physiological conditions. ...
Following the discovery that the amyloid precursor protein (APP) is the source of β-amyloid peptides (Aβ) that accumulate in Alzheimer’s disease (AD), structural analyses suggested that the holoprotein resembles a transmembrane receptor. Initial studies using reconstituted membranes demonstrated that APP can directly interact with the heterotrimeric G protein Gαo (but not other G proteins) via an evolutionarily G protein-binding motif in its cytoplasmic domain. Subsequent investigations in cell culture showed that antibodies against the extracellular domain of APP could stimulate Gαo activity, presumably mimicking endogenous APP ligands. In addition, chronically activating wild type APP or overexpressing mutant APP isoforms linked with familial AD could provoke Go-dependent neurotoxic responses, while biochemical assays using human brain samples suggested that the endogenous APP-Go interactions are perturbed in AD patients. More recently, several G protein-dependent pathways have been implicated in the physiological roles of APP, coupled with evidence that APP interacts both physically and functionally with Gαo in a variety of contexts. Work in insect models has demonstrated that the APP ortholog APPL directly interacts with Gαo in motile neurons, whereby APPL-Gαo signaling regulates the response of migratory neurons to ligands encountered in the developing nervous system. Concurrent studies using cultured mammalian neurons and organotypic hippocampal slice preparations have shown that APP signaling transduces the neuroprotective effects of soluble sAPPα fragments via modulation of the PI3K/Akt pathway, providing a mechanism for integrating the stress and survival responses regulated by APP. Notably, this effect was also inhibited by pertussis toxin, indicating an essential role for Gαo/i proteins. Unexpectedly, C-terminal fragments (CTFs) derived from APP have also been found to interact with Gαs, whereby CTF-Gαs signaling can promote neurite outgrowth via adenylyl cyclase/PKA-dependent pathways. These reports offer the intriguing perspective that G protein switching might modulate APP-dependent responses in a context-dependent manner. In this review, we provide an up-to-date perspective on the model that APP plays a variety of roles as an atypical G protein-coupled receptor in both the developing and adult nervous system, and we discuss the hypothesis that disruption of these normal functions might contribute to the progressive neuropathologies that typify AD.
... Our results demonstrate that single copy insertion of the human APP gene into the C. elegans genome leads to age-dependent degeneration of cholinergic neurons. While previous studies have not examined age-dependent neurodegeneration, multi-copy overexpression of the C. elegans ortholog of APP (apl-1) or human APP has been shown to cause behavioral dysfunction and partial lethality in C. elegans (Hornsten et al. 2007, Ewald et al. 2012. Importantly, the neurodegeneration we observed in transgenic APP worms was diminished by genetic manipulation of PGRMC1 or by pharmacological inhibition with SAS-0132 and JVW-1009. ...
Accumulating evidence suggests that modulating the sigma 2 receptor (Sig2R) can provide beneficial effects for neurodegenerative diseases. Herein, we report the identification of a novel class of Sig2R ligands and their cellular and in vivo activity in experimental models of Alzheimer's disease ( AD ). We report that SAS ‐0132 and DKR ‐1051, selective ligands of Sig2R, modulate intracellular Ca ²⁺ levels in human SK ‐N‐ SH neuroblastoma cells. The Sig2R ligands SAS ‐0132 and JVW ‐1009 are neuroprotective in a C. elegans model of amyloid precursor protein‐mediated neurodegeneration. Since this neuroprotective effect is replicated by genetic knockdown and knockout of vem‐1 , the ortholog of progesterone receptor membrane component‐1 ( PGRMC 1), these results suggest that Sig2R ligands modulate a PGRMC 1‐related pathway. Last, we demonstrate that SAS ‐0132 improves cognitive performance both in the Thy‐1 hAPP L ond/Swe+ transgenic mouse model of AD and in healthy wild‐type mice. These results demonstrate that Sig2R is a promising therapeutic target for neurocognitive disorders including AD .
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... Despite the substantial knowledge on the pathological features, the mechanism initiating or leading to the development of AD remains poorly understood. Two forms of AD, namely familial AD (FAD) and sporadic AD (SAD), are known to occur [39]. early onset FAD shows mutation in three genes: APP, presenilin 1 (PSeN1), and presenilin 2 (PSeN2). ...
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease and a worldwide health challenge. Different therapeutic approaches are being developed to reverse or slow the loss of affected neurons. Another plausible therapeutic way that may complement the studies is to increase the survival of existing neurons by mobilizing the existing neural stem/progenitor cells (NSPCs) — i.e. “induce their plasticity” — to regenerate lost neurons despite the existing pathology and unfavorable environment. However, there is controversy about how NSPCs are affected by the unfavorable toxic environment during AD. In this review, we will discuss the use of stem cells in neurodegenerative diseases and in particular how NSPCs affect the AD pathology and how neurodegeneration affects NSPCs. In the end of this review, we will discuss how zebrafish as a useful model organism with extensive regenerative ability in the brain might help to address the molecular programs needed for NSPCs to respond to neurodegeneration by enhanced neurogenesis
The lack of effective therapies that slow the progression of Alzheimer’s disease (AD) and related tauopathies highlights the need for a more comprehensive understanding of the fundamental cellular mechanisms underlying these diseases. Model organisms, including yeast, worms, and flies, provide simple systems with which to investigate the mechanisms of disease. The evolutionary conservation of cellular pathways regulating proteostasis and stress response in these organisms facilitates the study of genetic factors that contribute to, or protect against, neurodegeneration. Here, we review genetic modifiers of neurodegeneration and related cellular pathways identified in the budding yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster, focusing on models of AD and related tauopathies. We further address the potential of simple model systems to better understand the fundamental mechanisms that lead to AD and other neurodegenerative disorders.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13024-023-00664-x.
The amyloid-beta (Aβ) plaques found in Alzheimer's disease (AD) patients' brains contain collagens and are embedded extracellularly. Several collagens have been proposed to influence Aβ aggregate formation, yet their role in clearance is unknown. To investigate the potential role of collagens in forming and clearance of extracellular aggregates in vivo , we created a transgenic Caenorhabditis elegans strain that expresses and secretes human Aβ 1-42 . This secreted Aβ forms aggregates in two distinct places within the extracellular matrix. In a screen for extracellular human Aβ aggregation regulators, we identified different collagens to ameliorate or potentiate Aβ aggregation. We show that a disintegrin and metalloprotease ADM-2, an orthologue of ADAM9, reduces the load of extracellular Aβ aggregates. ADM-2 is required and sufficient to remove the extracellular Aβ aggregates. Thus, we provide in-vivo evidence of collagens essential for aggregate formation and metalloprotease participating in extracellular Aβ aggregate removal.
Neurodegenerative diseases (NDs) are a heterogeneous group of aging-associated ailments characterized by interrupting cellular proteostasic machinery and the misfolding of distinct proteins to form toxic aggregates in neurons. Neurodegenerative diseases, which include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and others, are becoming an increasing threat to human health worldwide. The degeneration and death of certain specific groups of neurons are the hallmarks of these diseases. Over the past decades, Caenorhabditis eleganshas beenwidely used as a transgenic model to investigate biological processes related to health and disease. The nematode Caenorhabditis elegans (C. elegans) has developed as a powerful tool for studying disease mechanisms due to its ease of genetic handling and instant cultivation while providing a whole-animal system amendable to several molecular and biochemical techniques. In this review, we elucidate the potential of C. elegans as a versatile platform for systematic dissection of the molecular basis of human disease, focusing on neurodegenerative disorders, and may help better our understanding of the disease mechanisms and search for new therapeutics for these devastating diseases.
Diverse models have been long explored to impel the progress of food science, while Caenorhabditis elegans (C. elegans) models applied in food toxicology and food function evaluation received vigorous advances recently. With multifaceted advantages such as short lifespan, apparent phenotypes and well-recognized genetic background, C. elegans is becoming a driving model organism in food science. In this review, we comprehensively reviewed the research progress of C. elegans models based on the literature published in past two decades, and systematically categorized the methods of C. elegans models’ construction, summarized the applications of nematodes in food toxicology, mainly involved in microbial contamination, food additives, pesticide residues, heavy metals, food packaging, and physical pollution. C. elegans models’ exploitations for functional foods assessments were also exemplified, and biological properties included anti-oxidant, anti-aging, anti-obesity, anti-glucotoxicity, anti-neurodegenerative and immunoregulation. Research gaps between present shortcomings and future orientations have been further discussed. We believed that this review would inspire ideas and strategies for further nematodes model’s exploration and multi-models-cooperation in food science.
The amyloid-beta (Aβ) plaques found in Alzheimer’s disease (AD) patients’ brains contain collagens and are embedded extracellularly. Several collagens have been proposed to influence Aβ aggregate formation, yet their role in clearance is unknown. To investigate the potential role of collagens in forming and clearance extracellular aggregates in vivo , we created a transgenic Caenorhabditis elegans strain that expresses and secretes human Aβ 1-42 . This secreted Aβ forms aggregates in two distinct places within the extracellular matrix. In a screen for extracellular human Aβ aggregation regulators, we identified different collagens to ameliorate or potentiate Aβ aggregation. We show that a disintegrin and metalloprotease ADM-2, an orthologue of ADAM9, reduces the load of extracellular Aβ aggregates. ADM-2 is required and sufficient to remove the extracellular Aβ aggregates. Thus, we provide in-vivo evidence of collagens essential for aggregate formation and metalloprotease participating in extracellular Aβ aggregate removal.
Highlights
Extracellular aggregates of amyloid beta are a hallmark of Alzheimer’s disease. Here we developed a novel C. elegans transgenic line that secretes human amyloid beta, which forms aggregates in the extracellular matrix (ECM). We show that ECM dynamics can disturb aggregation and that ADM-2, an ortholog of Human ADAM9, is involved in removing these extracellular aggregates.
Eukaryotes may experience oxygen deprivation under both physiological and pathological conditions. Because oxygen shortage leads to a reduction in cellular energy production, all eukaryotes studied so far conserve energy by suppressing their metabolism. However, the molecular physiology of animals that naturally and repeatedly experience anoxia is underexplored. One such animal is the marine nematode Laxus oneistus. It thrives, invariably coated by its sulfur-oxidizing symbiont Candidatus Thiosymbion oneisti, in anoxic sulfidic or hypoxic sand. Here, transcriptomics and proteomics showed that, whether in anoxia or not, L. oneistus mostly expressed genes involved in ubiquitination, energy generation, oxidative stress response, immune response, development, and translation. Importantly, ubiquitination genes were also highly expressed when the nematode was subjected to anoxic sulfidic conditions, together with genes involved in autophagy, detoxification and ribosome biogenesis. We hypothesize that these degradation pathways were induced to recycle damaged cellular components (mitochondria) and misfolded proteins into nutrients. Remarkably, when L. oneistus was subjected to anoxic sulfidic conditions, lectin and mucin genes were also upregulated, potentially to promote the attachment of its thiotrophic symbiont. Furthermore, the nematode appeared to survive oxygen deprivation by using an alternative electron carrier (rhodoquinone) and acceptor (fumarate), to rewire the electron transfer chain. On the other hand, under hypoxia, genes involved in costly processes (e.g., amino acid biosynthesis, development, feeding, mating) were upregulated, together with the worm’s Toll-like innate immunity pathway and several immune effectors (e.g., bactericidal/permeability-increasing proteins, fungicides). In conclusion, we hypothesize that, in anoxic sulfidic sand, L. oneistus upregulates degradation processes, rewires the oxidative phosphorylation and reinforces its coat of bacterial sulfur-oxidizers. In upper sand layers, instead, it appears to produce broad-range antimicrobials and to exploit oxygen for biosynthesis and development.
As human life expectancy increases, neurodegenerative diseases present a growing public health threat, for which there are currently few effective treatments. There is an urgent need to understand the molecular and genetic underpinnings of these disorders so new therapeutic targets can be identified. Here we present the argument that the simple nematode worm Caenorhabditis elegans is a powerful tool to rapidly study neurodegenerative disorders due to their short lifespan and vast array of genetic tools, which can be combined with characterization of conserved neuronal processes and behavior orthologous to those disrupted in human disease. We review how pre-existing C. elegans models provide insight into human neurological disease as well as an overview of current tools available to study neurodegenerative diseases in the worm, with an emphasis on genetics and behavior. We also discuss open questions that C. elegans may be particularly well suited for in future studies and how worms will be a valuable preclinical model to better understand these devastating neurological disorders.
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.
Eukaryotes may experience oxygen deprivation under both physiological and pathological conditions. Because oxygen shortage leads to a reduction in cellular energy production, all eukaryotes studied so far conserve energy by suppressing their metabolism. However, the molecular physiology of animals that naturally and repeatedly experience anoxia is underexplored. One such animal is the marine nematode Laxus oneistus . It thrives, invariably coated by its sulfur-oxidizing symbiont Candidatus Thiosymbion oneisti, in anoxic sulfidic or hypoxic sand. Here, transcriptomics and proteomics showed that, whether in anoxia or not, L. oneistus mostly expressed genes involved in ubiquitination, energy generation, oxidative stress response, immune response, development, and translation. Importantly, ubiquitination genes were also highly expressed when the nematode was subjected to anoxic sulfidic conditions, together with genes involved in autophagy, detoxification and ribosome biogenesis. We hypothesize that these degradation pathways were induced to recycle damaged cellular components (mitochondria) and misfolded proteins into nutrients. Remarkably, when L. oneistus was subjected to anoxic sulfidic conditions, lectin and mucin genes were also upregulated, potentially to promote the attachment of its thiotrophic symbiont. Furthermore, the nematode appeared to survive oxygen deprivation by using an alternative electron carrier (rhodoquinone) and acceptor (fumarate), to rewire the electron transfer chain. On the other hand, under hypoxia, genes involved in costly processes (e.g., amino acid biosynthesis, development, feeding, mating) were upregulated, together with the worm’s Toll-like innate immunity pathway and several immune effectors (e.g., Bacterial Permeability Increasing proteins, fungicides). In conclusion, we hypothesize that, in anoxic sulfidic sand, L. oneistus upregulates degradation processes, rewires oxidative phosphorylation and by reinforces its coat of bacterial sulfur-oxidizers. In upper sand layers, instead, it appears to produce broad-range antimicrobials and to exploit oxygen for biosynthesis and development.
Genetic and epidemiological studies have found that variations in the amyloid precursor protein ( APP ) and the apoliopoprotein E ( APOE ) genes represent major modifiers of the progressive neurodegeneration in Alzheimer's disease (AD). An extra copy of or gain-of-function mutations in APP correlate with early onset AD. Compared to the other variants ( APOE2 and APOE3 ), the ε4 allele of APOE ( APOE4 ) hastens and exacerbates early and late onset forms of AD. Convenient in vivo models to study how APP and APOE4 interact at the cellular and molecular level to influence neurodegeneration are lacking. Here, we show that the nematode C. elegans can model important aspects of AD including age-related, patterned neurodegeneration that is exacerbated by APOE4 . Specifically, we found that APOE4, but not APOE3 , acts with APP to hasten and expand the pattern of cholinergic neurodegeneration caused by APP . Molecular mechanisms underlying how APP and APOE4 synergize to kill some neurons while leaving others unaffected may be uncovered using this convenient worm model of neurodegeneration.
The nematode Caenorhabditis elegans offers unique advantages that enable a comprehensive delineation of the cellular and molecular mechanisms underlying devastating human pathologies such as stroke, ischemia and age-associated neurodegenerative disorders. Genetic models of human diseases that closely simulate several disease-related phenotypes have been established in the worm. These models allow the implementation of multidisciplinary approaches, in addition to large-scale genetic and pharmacological screenings, designed to elucidate the molecular mechanisms mediating pathogenesis and to identify targets and drugs for emergent therapeutic interventions. Such strategies have already provided valuable insights, highly relevant to human health and quality of life. This article considers the potential of C. elegans as a versatile platform for systematic dissection of the molecular basis of human disease, focusing on neurodegenerative disorders.
Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder that predominantly affects people aged over 65 years. AD is marked by cognitive deficits and memory problems that worsen with age and ultimately results in death. Pathology of AD includes aggregation of the amyloid beta peptide into extracellular plaques and the presence of hyperphosphorylated tau in intracellular neurofibrillary tangles. Given that many factors are involved in the disease along with the ability to study individual aspects of disease pathology under controlled conditions, several genetically tractable animal models have been developed. Despite years of research, treatments remain limited and many therapies that yield promising data in animal models fail to translate it in humans. Here, we discuss the use of a highly versatile Drosophila melanogaster (aka fruit fly) model to study AD. The genetic machinery is conserved from fly to humans. The Drosophila eye has proved to be a genetically tractable model to study neurodegenerative disorders and for genetic and chemical screens. We highlight the utility of modeling AD by expressing human Aβ42 in the developing Drosophila retina. This system has been used recently to uncover new factors involved in the pathological activation of cell death pathways in AD. We discuss these findings and their role in the search for new disease treatments.
The drug-discovery process is expensive and lengthy, and has been causing a rapid increase in the global health care cost. Despite extensive efforts, many human diseases still lack a cure. To improve the outcomes, there is a growing need to implement novel approaches into the early stages of the drug-discovery pipeline. A specific such effort has focused on the development of novel disease models such as cellular models (genetically modified cell lines, spheroids, and organoids) and whole-animal models (small animal models and genetically modified large animal models). The whole-animal screens are advantageous as they can provide system-level information, off-target effects, complete absorption, distribution, metabolism, excretion, and toxicity architectures, and early in vivo toxicity, which help to prioritize compounds before using them for human trials. Such multivariate analysis helps to improve the translational potential of drug compounds. Drug testing in large animals is expensive and time consuming. A solution is small animal models that have simplified biological system with intact physiology and sufficient homology with human genes. In recent times, many such models have constantly been developed and tested to identify new disease mechanisms. Caenorhabditis elegans is one such small animal model that has been considered for large-scale drug testing. In this review, we will discuss the current state-of-the-art technologies, including two platforms developed in my group that have enabled high-throughput and high-content screening using C. elegans disease models.
The societal burden presented by Alzheimer’s disease warrants both innovative and expedient means by which its underlying molecular causes can be both identified and mechanistically exploited to discern novel therapeutic targets and strategies. The conserved characteristics, defined neuroanatomy and advanced technological application of Caenorhabditis elegans render this metazoan an unmatched tool for probing neurotoxic factors. In addition, its short lifespan and importance in the field of aging make it an ideal organism for modeling age-related neurodegenerative disease. As such, this nematode system has demonstrated its value in predicting functional modifiers of human neurodegenerative disorders. Here, we review how C. elegans has been utilized to model Alzheimer’s disease. Specifically, we present how the causative neurotoxic peptides, amyloid-β and tau, contribute to disease-like neurodegeneration in C. elegans and how they translate to human disease. Furthermore, we describe how a variety of transgenic animal strains, each with distinct utility, have been used to identify both genetic and pharmacological modifiers of toxicity in C. elegans. As technological advances improve the prospects for intervention, the rapidity, unparalleled accuracy, and scale that C. elegans offers researchers for defining functional modifiers of neurodegeneration should speed the discovery of improved therapies for Alzheimer's disease.
The amyloid precursor protein (APP) is a receptor-like membrane protein. Although APP processing and β-amyloid production play a central role in Alzheimer’s disease (AD) pathogenesis, the physiological function of APP remains elusive. Here, we identify APP as a novel receptor for Slit that mediates axon guidance and neural circuit formation. APP deficiency abolishes the Slit repulsive effect in a 3D olfactory explant culture, consistent with its callosal projection deficit in vivo and reminiscent of Slit loss. Inactivation of APP ortholog APL-1 in Caenorhabditis elegans results in pioneer axon mistargeting and genetic analysis places APL-1 in the SLT-1 (Slit)/SAX-3 (Robo) repulsive pathway. Slit binds to APP through the E1 domain, which triggers APP ectodomain shedding and recruitment of the intracellular FE65 and Pak1 complex and associated Rac1 GTPase activation. Our study establishes APP as a novel receptor for Slit ligand mediating axon guidance and neural circuit formation.
Neurodegenerative diseases, such as Alzheimer's disease (AD), are multifactorial diseases involving age, genetics, and environment. As such, the model in which one examines disease biology and potential therapeutics should incorporate these three salient features. In both cell culture systems and rodent models, both environment contributions and genetic background can be easily assessed. However, studying the effects of age is either not possible (cell culture) or expensive (rodents), especially for drug discovery. Additionally, the complex mammalian brain is not the easiest to image in vivo for AD pathology hallmarks. For these reasons, simple genetic model organisms can be utilized. Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio have long been used in research; taking advantage of short lifespans, well-characterized genetics, and inexpensive maintenance for environmental and pharmacology studies. In this chapter, the benefits of these organisms for AD research will be discussed, including available transgenics and potential for use in pharmaceutical testing.
Next generation drug screening could benefit greatly from in vivo studies, using small animal models such as Caenorhabditis elegans for hit identification and lead optimization. Current in vivo assays can operate either at low throughput with high resolution or with low resolution at high throughput. To enable both high-throughput and high-resolution imaging of C. elegans, we developed an automated microfluidic platform. This platform can image 15 z-stacks of ∼4,000 C. elegans from 96 different populations using a large-scale chip with a micron resolution in 16 min. Using this platform, we screened ∼100,000 animals of the poly-glutamine aggregation model on 25 chips. We tested the efficacy of ∼1,000 FDA-approved drugs in improving the aggregation phenotype of the model and identified four confirmed hits. This robust platform now enables high-content screening of various C. elegans disease models at the speed and cost of in vitro cell-based assays.
Significance statement:
Synaptic dysfunction and cognitive decline are early hallmark features of Alzheimer's disease. Thus, it is essential to elucidate the in vivo function(s) of APP at the synapse. At present, it is unknown whether APP family proteins function as cell surface receptors, or mainly via shedding of their secreted ectodomains, such as neurotrophic APPsα. Here, to dissect APP functional domains, we used APP mutant mice lacking the last 15 amino acids that were crossed onto an APLP2-KO background. These APPΔCT15-DM mice showed defects in neuromuscular morphology and function. Synaptic deficits in the CNS included impairments of synaptic plasticity, spatial learning, and memory. Collectively, this indicates that multiple APP domains, including the C-terminus, are required for normal nervous system function.
Age-associated neurodegenerative disorders such as Alzheimer’s disease are a major public health challenge, due to the demographic increase in the proportion of older individuals in society. However, the relatively few currently approved drugs for these conditions provide only symptomatic relief. A major goal of neurodegeneration research is therefore to identify potential new therapeutic compounds that can slow or even reverse disease progression, either by impacting directly on the neurodegenerative process or by activating endogenous physiological neuroprotective mechanisms that decline with ageing. This requires model systems that can recapitulate key features of human neurodegenerative diseases that are also amenable to compound screening approaches. Mammalian models are very powerful, but are prohibitively expensive for high-throughput drug screens. Given the highly conserved neurological pathways between mammals and invertebrates, Caenorhabditis elegans has emerged as a powerful tool for neuroprotective compound screening. Here we describe how C. elegans has been used to model various human ageing-associated neurodegenerative diseases and provide an extensive list of compounds that have therapeutic activity in these worm models and so may have translational potential.
The amyloid precursor protein (APP) involved in Alzheimer's disease is a member of a larger gene family including amyloid precursor-like proteins APLP1 and APLP2. We generated and examined the phenotypes of mice lacking individual or all possible combinations of APP family members to assess potential functional redundancies within the gene family. Mice deficient for the nervous system-specific APLP1 protein showed a postnatal growth deficit as the only obvious abnormality. In contrast to this minor phenotype, APLP2 ⁻ /− /APLP1 ⁻ /− and APLP2 ⁻ /− /APP ⁻ /− mice proved lethal early postnatally. Surprisingly, APLP1 ⁻ /− /APP ⁻ /− mice were viable, apparently normal, and showed no compensatory upregulation of APLP2 expression. These data indicate redundancy between APLP2 and both other family members and corroborate a key physiological role for APLP2. This view gains further support by the observation that APLP1 ⁻ /− /APP ⁻ /− /APLP2 +/− mice display postnatal lethality. In addition, they provide genetic evidence for at least some distinct physiological roles of APP and APLP2 by demonstrating that combinations of single knock-outs with the APLP1 mutation resulted in double mutants of clearly different phenotypes, being either lethal, or viable. None of the lethal double mutants displayed, however, obvious histopathological abnormalities in the brain or any other organ examined. Moreover, cortical neurons from single or combined mutant mice showed unaltered survival rates under basal culture conditions and unaltered susceptibility to glutamate excitotoxicity in vitro .
The rol-6 gene is one of the more than 40 loci in Caenorhabditis elegans that primarily affect organismal morphology. Certain
mutations in the rol-6 gene produce animals that have the right roller phenotype, i.e., they are twisted into a right-handed
helix. The rol-6 gene interacts with another gene that affects morphology, sqt-1; a left roller allele of sqt-1 acts as a
dominant suppressor of a right roller allele of rol-6. The sqt-1 gene has previously been shown to encode a collagen. We isolated
and sequenced the rol-6 gene and found that it also encodes a collagen. The rol-6 gene was identified by physical mapping
of overlapping chromosomal deficiencies that cover the gene and by identification of an allele-specific restriction site alteration.
The amino acid sequence of the collagen encoded by rol-6 is more similar to that of the sqt-1 collagen than to any of the
other ten C. elegans cuticle collagen sequences compared. The locations of cysteine residues flanking the Gly-X-Y repeat regions
of rol-6 and sqt-1 are identical, but differ from those in the other collagens. The sequence similarities between rol-6 and
sqt-1 indicate that they represent a new collagen subfamily in C. elegans. These findings suggest that these two collagens
physically interact, possibly explaining the genetic interaction seen between the rol-6 and sqt-1 genes.
We have isolated genomic and cDNA clones for a Drosophila gene resembling the human beta-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human beta-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.
In several pedigrees of early onset familial Alzheimer's disease (FAD), point mutations in the beta-amyloid precursor protein (APP) gene are genetically linked to the disease. This finding implicates APP in the pathogenesis of Alzheimer's disease in these individuals. To understand the in vivo function of APP and its processing, we have generated an APP-null mutation in mice. Homozygous APP-deficient mice were viable and fertile. However, the mutant animals weighed 15%-20% less than age-matched wild-type controls. Neurological evaluation showed that the APP-deficient mice exhibited a decreased locomotor activity and forelimb grip strength, indicating a compromised neuronal or muscular function. In addition, four out of six homozygous mice showed reactive gliosis at 14 weeks of age, suggesting an impaired neuronal function as a result of the APP-null mutation.
During Caenorhabditis elegans vulval development, an inductive signal from the anchor cell stimulates three of the six vulval precursor cells (VPCs) to adopt vulval rather than nonvulval epidermal fates. Genes necessary for this induction include the lin-3 growth factor, the let-23 receptor tyrosine kinase, and let-60 ras. lin-15 is a negative regulator of this inductive pathway. In lin-15 mutant animals, all six VPCs adopt vulval fates, even in the absence of inductive signal. Previous genetic studies suggested that lin-15 is a complex locus with two independently mutable activities, A and B. We have cloned the lin-15 locus by germline transformation and find that it encodes two nonoverlapping transcripts that are transcribed in the same direction. The downstream transcript encodes the lin-15A function; the upstream transcript encodes the lin-15B function. The predicted lin-15A and lin-15B proteins are novel and hydrophilic. We have identified a molecular null allele of lin-15 and have used it to analyze the role of lin-15 in the signaling pathway. We find that lin-15 acts upstream of let-23 and in parallel to the inductive signal.
Four Caenorhabditis elegans genes encode muscle-type specific myosin heavy chain isoforms: myo-1 and myo-2 are expressed in the pharyngeal muscles; unc-54 and myo-3 are expressed in body wall muscles. We have used transformation-rescue and lacZ fusion assays to determine sequence requirements for regulated myosin gene expression during development. Multiple tissue-specific activation elements are present for all four genes. For each of the four genes, sequences upstream of the coding region are tissue-specific promoters, as shown by their ability to drive expression of a reporter gene (lacZ) in the appropriate muscle type. Each gene contains at least one additional tissue-specific regulatory element, as defined by the ability to enhance expression of a heterologous promoter in the appropriate muscle type. In rescue experiments with unc-54, two further requirements apparently independent of tissue specificity were found: sequences within the 3' non-coding region are essential for activity while an intron near the 5' end augments expression levels. The general intron stimulation is apparently independent of intron sequence, indicating a mechanistic effect of splicing. To further characterize the myosin gene promoters and to examine the types of enhancer sequences in the genome, we have initiated a screen of C. elegans genomic DNA for fragments capable of enhancing the myo-2 promoter. The properties of enhancers recovered from this screen suggest that the promoter is limited to muscle cells in its ability to respond to enhancers.
Rab molecules regulate vesicular trafficking in many different exocytic and endocytic transport pathways in eukaryotic cells. In neurons, rab3 has been proposed to play a crucial role in regulating synaptic vesicle release. To elucidate the role of rab3 in synaptic transmission, we isolated and characterized Caenorhabditis elegans rab-3 mutants. Similar to the mouse rab3A mutants, these mutants survived and exhibited only mild behavioral abnormalities. In contrast to the mouse mutants, synaptic transmission was perturbed in these animals. Extracellular electrophysiological recordings revealed that synaptic transmission in the pharyngeal nervous system was impaired. Furthermore, rab-3 animals were resistant to the acetylcholinesterase inhibitor aldicarb, suggesting that cholinergic transmission was generally depressed. Last, synaptic vesicle populations were redistributed in rab-3 mutants. In motor neurons, vesicle populations at synapses were depleted to 40% of normal levels, whereas in intersynaptic regions of the axon, vesicle populations were elevated. On the basis of the morphological defects at neuromuscular junctions, we postulate that RAB-3 may regulate recruitment of vesicles to the active zone or sequestration of vesicles near release sites.
Synaptobrevins are vesicle-associated proteins implicated in neurotransmitter release by both biochemical studies and perturbation experiments that use botulinum toxins. To test these models in vivo, we have isolated and characterized the first synaptobrevin mutants in metazoans and show that neurotransmission is severely disrupted in mutant animals. Mutants lacking snb-1 die just after completing embryogenesis. The dying animals retain some capability for movement, although they are extremely uncoordinated and incapable of feeding. We also have isolated and characterized several hypomorphic snb-1 mutants. Although fully viable, these mutants exhibit a variety of behavioral abnormalities that are consistent with a general defect in the efficacy of synaptic transmission. The viable mutants are resistant to the acetylcholinesterase inhibitor aldicarb, indicating that cholinergic transmission is impaired. Extracellular recordings from pharyngeal muscle also demonstrate severe defects in synaptic transmission in the mutants. The molecular lesions in the hypomorphic alleles reside on the hydrophobic face of a proposed amphipathic-helical region implicated biochemically in interacting with the t-SNAREs syntaxin and SNAP-25. Finally, we demonstrate that double mutants lacking both the v-SNAREs synaptotagmin and snb-1 are phenotypically similar to snb-1 mutants and less severe than syntaxin mutants. Our work demonstrates that synaptobrevin is essential for viability and is required for functional synaptic transmission. However, our analysis also suggests that transmitter release is not completely eliminated by removal of either one or both v-SNAREs.
Biochemical and molecular mechanisms of neuronal cell death are currently an area of intense research. It is well documented that the lumbar spinal motoneurons of the chick embryo undergo a period of naturally occurring programmed cell death (PCD) requiring new gene expression and activation of caspases. To identify genes that exhibit changed expression levels in dying motoneurons, we used a PCR-based subtractive hybridization protocol to identify messages uniquely expressed in motoneurons deprived of trophic support as compared with their healthy counterparts. We report that one upregulated message in developing motoneurons undergoing cell death is the mRNA for amyloid precursor protein (APP). Increased levels of APP and beta-amyloid protein are also detected within dying motoneurons. The predicted peptide sequence of APP indicates two potential cleavage sites for caspase-3 (CPP-32), a caspase activated in dying motoneurons. When peptide inhibitors of caspase-3 are administered to motoneurons destined to undergo PCD, decreased levels of APP protein and greatly reduced beta-amyloid production are observed. Furthermore, we show that APP is cleaved by caspase-3. Our results suggest that differential gene expression results in increased levels of APP, providing a potential substrate for one of the cell death-activated caspases that may ultimately cause the demise of the cell. These results, combined with information on the toxic role of APP and its proteolytic by-product beta-amyloid, in the neurodegenerative disease Alzheimer's, suggest that events of developmental PCD may be reactivated in early stages of pathological neurodegeneration.
The importance of the amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease (AD) became apparent through the identification of distinct mutations in the APP gene, causing early onset familial AD with the accumulation of a 4-kDa peptide fragment (betaA4) in amyloid plaques and vascular deposits. However, the physiological role of APP is still unclear. In this work, Drosophila melanogaster is used as a model system to analyze the function of APP by expressing wild-type and various mutant forms of human APP in fly tissue culture cells as well as in transgenic fly lines. After expression of full-length APP forms, secretion of APP but not of betaA4 was observed in both systems. By using SPA4CT, a short APP form in which the signal peptide was fused directly to the betaA4 region, transmembrane domain, and cytoplasmic tail, we observed betaA4 release in flies and fly-tissue culture cells. Consequently, we showed a gamma-secretase activity in flies. Interestingly, transgenic flies expressing full-length forms of APP have a blistered-wing phenotype. As the wing is composed of interacting dorsal and ventral epithelial cell layers, this phenotype suggests that human APP expression interferes with cell adhesion/signaling pathways in Drosophila, independently of betaA4 generation.
We have examined the trafficking and metabolism of the beta-amyloid precursor protein (APP), an APP homolog (APLP1), and TrkB in neurons that lack PS1. We report that PS1-deficient neurons fail to secrete Abeta, and that the rate of appearance of soluble APP derivatives in the conditioned medium is increased. Remarkably, carboxyl-terminal fragments (CTFs) derived from APP and APLP1 accumulate in PS1-deficient neurons. Hence, PS1 plays a role in promoting intramembrane cleavage and/or degradation of membrane-bound CTFs. Moreover, the maturation of TrkB and BDNF-inducible TrkB autophosphorylation is severely compromised in neurons lacking PS1. We conclude that PS1 plays an essential role in modulating trafficking and metabolism of a selected set of membrane and secretory proteins in neurons.
Neuronal cell death, neurofibrillary tangles, and amyloid beta peptide (Abeta) deposition depict Alzheimer's disease (AD) pathology, but neuronal loss correlates best with dementia. We have shown that increased production of Abeta is a consequence of neuronal apoptosis, suggesting that apoptosis activates proteases involved in amyloid precursor protein (APP) processing. Here, we investigate key effectors of cell death, caspases, in human neuronal apoptosis and APP processing. We find that caspase-6 is activated and responsible for neuronal apoptosis by serum deprivation. Caspase-6 activity precedes the time of commitment to neuronal apoptosis by 10 h, indicating possible activity without subsequent apoptosis. Inhibition of caspase-6 activity prevents serum deprivation-mediated increase of Abeta. Caspase-6 directly cleaves APP at the C terminus and generates a C-terminal fragment of 3 kDa (Capp3) and an Abeta-containing 6.5-kDa fragment, Capp6.5, that increases in serum-deprived neurons. A pulse-chase experiment reveals a precursor-product relationship between Capp6.5, intracellular Abeta, and secreted Abeta, indicating a potential alternate amyloidogenic pathway. Caspase-6 proenzyme is present in adult human brain tissue, and the p10 active caspase-6 fragment is detected in AD brain tissue. These results indicate a possible alternate pathway for APP amyloidogenic processing in human neurons and a potential implication for this pathway in the neuronal demise of AD.
Although abnormal processing of beta-amyloid precursor protein (APP) has been implicated in the pathogenic cascade leading to Alzheimer's disease, the normal function of this protein is poorly understood. To gain insight into APP function, we used a molecular-genetic approach to manipulate the structure and levels of the Drosophila APP homolog APPL. Wild-type and mutant forms of APPL were expressed in motoneurons to determine the effect of APPL at the neuromuscular junction (NMJ). We show that APPL was transported to motor axons and that its overexpression caused a dramatic increase in synaptic bouton number and changes in synapse structure. In an Appl null mutant, a decrease in the number of boutons was found. Examination of NMJs in larvae overexpressing APPL revealed that the extra boutons had normal synaptic components and thus were likely to form functional synaptic contacts. Deletion analysis demonstrated that APPL sequences responsible for synaptic alteration reside in the cytoplasmic domain, at the internalization sequence GYENPTY and a putative G(o)-protein binding site. To determine the likely mechanisms underlying APPL-dependent synapse formation, hyperexcitable mutants, which also alter synaptic growth at the NMJ, were examined. These mutants with elevated neuronal activity changed the distribution of APPL at synapses and partially suppressed APPL-dependent synapse formation. We propose a model by which APPL, in conjunction with activity-dependent mechanisms, regulates synaptic structure and number.
The Alzheimer's disease amyloid protein precursor (APP) gene is part of a multi-gene super-family from which sixteen homologous amyloid precursor-like proteins (APLP) and APP species homologues have been isolated and characterised. Comparison of exon structure (including the uncharacterised APL-1 gene), construction of phylogenetic trees, and analysis of the protein sequence alignment of known homologues of the APP super-family were performed to reconstruct the evolution of the family and to assess the functional significance of conserved protein sequences between homologues. This analysis supports an adhesion function for all members of the APP super family, with specificity determined by those sequences which are not conserved between APLP lineages, and provides evidence for an increasingly complex APP superfamily during evolution. The analysis also suggests that Drosophila APPL and Caenorhabditis elegans APL-1 may be a fourth APLP lineage indicating that these proteins, while not functional homologues of human APP, are similarly likely to regulate cell adhesion. Furthermore, the betaA4 sequence is highly conserved only in APP orthologues, strongly suggesting this sequence is of significant functional importance in this lineage.
The amyloid precursor protein (APP) involved in Alzheimer's disease is a member of a larger gene family including amyloid precursor-like proteins APLP1 and APLP2. We generated and examined the phenotypes of mice lacking individual or all possible combinations of APP family members to assess potential functional redundancies within the gene family. Mice deficient for the nervous system-specific APLP1 protein showed a postnatal growth deficit as the only obvious abnormality. In contrast to this minor phenotype, APLP2(-/-)/APLP1(-/-) and APLP2(-/-)/APP(-/-) mice proved lethal early postnatally. Surprisingly, APLP1(-/-)/APP(-/-) mice were viable, apparently normal, and showed no compensatory upregulation of APLP2 expression. These data indicate redundancy between APLP2 and both other family members and corroborate a key physiological role for APLP2. This view gains further support by the observation that APLP1(-/-)/APP(-/-)/APLP2(+/-) mice display postnatal lethality. In addition, they provide genetic evidence for at least some distinct physiological roles of APP and APLP2 by demonstrating that combinations of single knock-outs with the APLP1 mutation resulted in double mutants of clearly different phenotypes, being either lethal, or viable. None of the lethal double mutants displayed, however, obvious histopathological abnormalities in the brain or any other organ examined. Moreover, cortical neurons from single or combined mutant mice showed unaltered survival rates under basal culture conditions and unaltered susceptibility to glutamate excitotoxicity in vitro.
We analyzed the mechanism of axonal transport of the amyloid precursor protein (APP), which plays a major role in the development of Alzheimer's disease. Coimmunoprecipitation, sucrose gradient, and direct in vitro binding demonstrated that APP forms a complex with the microtubule motor, conventional kinesin (kinesin-I), by binding directly to the TPR domain of the kinesin light chain (KLC) subunit. The estimated apparent Kd for binding is 15-20 nM, with a binding stoichiometry of two APP per KLC. In addition, association of APP with microtubules and axonal transport of APP is greatly decreased in a gene-targeted mouse mutant of the neuronally enriched KLC1 gene. We propose that one of the normal functions of APP may be as a membrane cargo receptor for kinesin-I and that KLC is important for kinesin-I-driven transport of APP into axons.
PS1 deficiency and expression of PS1 with substitutions of two conserved transmembrane aspartate residues ("PS1 aspartate variants") leads to the reduction of Abeta peptide secretion and the accumulation of amyloid precursor protein (APP) C-terminal fragments. To define the nature of the "dominant negative" effect of the PS1 aspartate variants, we stably expressed PS1 harboring aspartate to alanine substitutions at codons 257 (D257A) or 385 (D385A), singly or in combination (D257A/D385A), in mouse neuroblastoma, N2a cells. Expression of the PS1 aspartate variants resulted in marked accumulation of intracellular and cell surface APP C-terminal fragments. While expression of the D385A PS1 variant reduced the levels of secreted Abeta peptides, we now show that neither the PS1 D257A nor D257A/D385A variants impair Abeta production. Surprisingly, the stability of both immature and mature forms of APP is dramatically elevated in cells expressing PS1 aspartate variants, commensurate with an increase in the cell surface levels of APP. These findings lead us to conclude that the stability and trafficking of APP can be profoundly modulated by coexpression of PS1 with mutations at aspartate 257 and aspartate 385.
Neurodegenerative diseases can be genetic or sporadic in origin. Genetic analysis has changed the study of the pathogenesis of these disorders by showing the causative functions of rare mutations. Yet, in the most common age-associated neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, the causes of neurodegeneration remain to be clarified. The observations that presenilin modulates proteolysis and turnover of several signaling molecules have led to speculations that pathways that are important in development may contribute to neurodegeneration. In this article, the possibility that these presenilin-regulated molecules may contribute to neurodegeneration is reviewed.
The Alzheimer's disease beta-amyloid precursor protein (APP) is a member of a larger gene family that includes the amyloid precursor-like proteins, termed APLP1 and APLP2. We previously documented that APLP2-/-APLP1-/- and APLP2-/-APP-/- mice die postnatally, while APLP1-/-APP-/- mice and single mutants were viable. We now report that mice lacking all three APP/APLP family members survive through embryonic development, and die shortly after birth. In contrast to double-mutant animals with perinatal lethality, 81% of triple mutants showed cranial abnormalities. In 68% of triple mutants, we observed cortical dysplasias characterized by focal ectopic neuroblasts that had migrated through the basal lamina and pial membrane, a phenotype that resembles human type II lissencephaly. Moreover, at E18.5 triple mutants showed a partial loss of cortical Cajal Retzius (CR) cells, suggesting that APP/APLPs play a crucial role in the survival of CR cells and neuronal adhesion. Collectively, our data reveal an essential role for APP family members in normal brain development and early postnatal survival.
Proteolytic processing of the amyloid-beta precursor protein (APP) generates the Abeta amyloid peptide of Alzheimer's disease. The biological function of APP itself remains, however, unclear. In the current review, we study in detail the different subdomains of APP and try to assign functional significance to particular structures identified in the protein.
The rol-6 gene is one of the more than 40 loci in Caenorhabditis elegans that primarily affect organismal morphology. Certain mutations in the rol-6 gene produce animals that have the right roller phenotype, i.e., they are twisted into a right-handed helix. The rol-6 gene interacts with another gene that affects morphology, sqt-1; a left roller allele of sqt-1 acts as a dominant suppressor of a right roller allele of rol-6. The sqt-1 gene has previously been shown to encode a collagen. We isolated and sequenced the rol-6 gene and found that it also encodes a collagen. The rol-6 gene was identified by physical mapping of overlapping chromosomal deficiencies that cover the gene and by identification of an allele-specific restriction site alteration. The amino acid sequence of the collagen encoded by rol-6 is more similar to that of the sqt-1 collagen than to any of the other ten C. elegans cuticle collagen sequences compared. The locations of cysteine residues flanking the Gly-X-Y repeat regions of rol-6 and sqt-1 are identical, but differ from those in the other collagens. The sequence similarities between rol-6 and sqt-1 indicate that they represent a new collagen subfamily in C. elegans. These findings suggest that these two collagens physically interact, possibly explaining the genetic interaction seen between the rol-6 and sqt-1 genes.
Protein–protein interactions are a molecular basis for the structural and functional organization within cells. They are mediated by a growing number of protein modules that bind peptide targets. Alterations in binding affinities can have serious consequences for some essential cellular processes. The three proteins identified to have mutations in their corresponding genes leading to presenile Alzheimer dementia (AD)—the amyloid precursor protein (APP) and presenilin 1 and 2—all interact with other proteins. The nature and function of these interacting proteins may contribute to elucidating the proper physiological functions of the AD proteins. APP-interacting proteins are pointing toward a function of APP in cell adhesion and neurite outgrowth and signaling. Proteins interacting with the presenilins however are more diverse in nature linking presenilin function to regulation in different signaling pathways including Wnt and Notch but also in apoptosis and Ca2+ homeostasis. Further research however is still needed to delineate the exact functional relevance of these interactions with respect to the physiological functions of the AD proteins in particular and the contribution of these proteins to AD pathogenesis in general.
The growth and reproduction of the small nematode Caenorhabditis elegans has been studied using an electronic nematode counter recently developed in our laboratory. At 20°C, the usual growth temperature, size increases in a smooth sigmoidal manner with time, linear growth being most rapid around the time of the fourth molt and nearly ceasing by the end of the period of egg-laying. Growth of populations is highly synchronous; the small residual size heterogeneity is maximal at about the time of maximal growth. The four molts do not involve major interruption of growth, but they do entail slight shape changes (elongating upon escape from the old cuticle). Egg-laying begins shortly after the fourth molt, the rate rising rapidly at first, then more gradually to a peak followed by a relatively rapid fall. Comparable measurements at 16 and 25°C establish that these are acceptable limit temperatures for work with temperature-sensitive mutants, although egg yield is somewhat reduced and the kinetics of egg-laying are altered at 25°C. Developmental chronologies for all three temperatures are presented.
Drosophila amyloid precursor protein-like (Appl) gene encodes a protein product (APPL) similar to beta-amyloid precursor protein (APP) associated with Alzheimer's disease. To understand the in vivo function of APPL protein, we have generated flies deleted for the Appl gene. These flies are viable, fertile, and morphologically normal, yet they exhibit subtle behavioral deficits. We show that a fast phototaxis defect in Appl- flies is partially rescued by transgenes expressing the wild-type, but not a mutant, APPL protein. We further demonstrate a functional homology between APPL and APP, since transgenes expressing human APP show a similar level of rescue as transgenes expressing fly APPL.
Alzheimer's disease is characterized by a widespread functional disturbance of the human brain. Fibrillar amyloid proteins are deposited inside neurons as neurofibrillary tangles and extracellularly as amyloid plaque cores and in blood vessels. The major protein subunit (A4) of the amyloid fibril of tangles, plaques and blood vessel deposits is an insoluble, highly aggregating small polypeptide of relative molecular mass 4,500. The same polypeptide is also deposited in the brains of aged individuals with trisomy 21 (Down's syndrome). We have argued previously that the A4 protein is of neuronal origin and is the cleavage product of a larger precursor protein. To identify this precursor, we have now isolated and sequenced an apparently full-length complementary DNA clone coding for the A4 polypeptide. The predicted precursor consists of 695 residues and contains features characteristic of glycosylated cell-surface receptors. This sequence, together with the localization of its gene on chromosome 21, suggests that the cerebral amyloid deposited in Alzheimer's disease and aged Down's syndrome is caused by aberrant catabolism of a cell-surface receptor.
We have purified and characterized the cerebral amyloid protein that forms the plaque core in Alzheimer disease and in aged individuals with Down syndrome. The protein consists of multimeric aggregates of a polypeptide of about 40 residues (4 kDa). The amino acid composition, molecular mass, and NH2-terminal sequence of this amyloid protein are almost identical to those described for the amyloid deposited in the congophilic angiopathy of Alzheimer disease and Down syndrome, but the plaque core proteins have ragged NH2 termini. The shared 4-kDa subunit indicates a common origin for the amyloids of the plaque core and of the congophilic angiopathy. There are superficial resemblances between the solubility characteristics of the plaque core and some of the properties of scrapie infectivity, but there are no similarities in amino acid sequences between the plaque core and scrapie polypeptides.
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.
A purified protein derived from the twisted beta-pleated sheet fibrils in cerebrovascular amyloidosis associated with Alzheimer's disease has been isolated by Sephadex G-100 column chromatography with 5 M guanidine-HC1 in 1 N acetic acid and by high performance liquid chromatography. Amino acid sequence analysis and a computer search reveals this protein to have no homology with any protein sequenced thus far. This protein may be derived from a unique serum precursor which may provide a diagnostic test for Alzheimer's disease and a means to understand its pathogenesis.
The secreted form of beta-amyloid precursor protein (APP) containing the Kunitz proteinase inhibitor (KPI) domain, also called protease nexin II, is internalized and degraded by cells. We show that the low density lipoprotein (LDL) receptor-related protein (LRP) is responsible for the endocytosis of secreted APP. APPs770 degradation is inhibited by an LRP antagonist called the receptor-associated protein (RAP) and by LRP antibodies and is greatly diminished in fibroblasts genetically deficient in LRP. APPs695, which lacks the KPI domain, is a poor LRP ligand. Since LRP also binds apolipoprotein E (apoE)-enriched lipoproteins and inheritance of the epsilon 4 allele of the apoE gene is a risk factor for Alzheimer's disease (AD), these data link in a single metabolic pathway two molecules strongly implicated in the pathophysiology of AD.
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.
In Caenorhabditis elegans, the terminal differentiation of the hypodermal cells occurs at the larval-to-adult molt, and is characterized in part by the formation of a morphologically distinct adult cuticle. The timing of this event is controlled by a pathway of heterochronic genes that includes the relatively direct regulatory gene, lin-29, and upstream genes lin-4, lin-14 and lin-28. Using northern analysis to detect endogenous collagen mRNA levels and collagen/lacZ reporter constructs to monitor collagen transcriptional activity, we show that the stage-specific switch from larval cuticle to adult cuticle correlates with the transcriptional activation of adult-specific collagen genes and repression of larval-specific collagen genes. Heterochronic mutations that cause precocious formation of adult cuticle also cause precocious transcription of the adult-specific collagen genes, col-7 and col-19; heterochronic mutations that prevent the switch to adult cuticle cause continued expression of the larval collagen gene, col-17, in adults and prevent adult-specific activation of col-7 or col-19. A 235 bp segment of col-19 5' sequences is sufficient to direct the adult-specific expression of a col-19/lacZ reporter gene in hypodermal cells. These findings indicate that the heterochronic gene pathway regulates the timing of hypodermal cell terminal differentiation by regulating larval- and adult-specific gene expression, perhaps by the direct action of lin-29.
The major component of senile plaques found in the brains of Alzheimer disease patients is the beta-amyloid peptide, which is derived from a larger amyloid precursor protein (APP). Recently, a number of APP and APP-related proteins have been identified in different organisms and constitute the family of APP proteins. We have isolated several cDNAs encoding an APP-related protein in the nematode Caenorhabditis elegans and have designated the corresponding gene as apl-1. The apl-1 transcripts undergo two forms of posttranscriptional modification: trans-splicing and alternative polyadenylylation. In vitro translation of an apl-1 cDNA results in a protein of approximately the expected size. Similar to the Drosophila, human, and mouse APP-related proteins, APL-1 does not appear to contain the beta-amyloid peptide. Because APP-related proteins seem to be conserved through evolution, the apl-1 gene from C. elegans should be important for determining the normal function of human APP.
To understand how genotype determines the phenotype of the animal Caenorhabditis elegans, one ideally needs to know the complete sequence of the genome and the contribution of genes to phenotype, which requires an efficient strategy for reverse genetics. We here report that the Tc1 transposon induces frequent deletions of flanking DNA, apparently resulting from Tc1 excision followed by imprecise DNA repair. We use this to inactivate genes in two steps. (i) We established a frozen library of 5000 nematode lines mutagenized by Tc1 insertion, from which insertion mutants of genes of interest can be recovered. Their address within the library is determined by PCR. (ii) Animals are then screened, again by PCR, to detect derivatives in which Tc1 and 1000-2000 base pairs of flanking DNA are deleted, and thus a gene of interest is inactivated. We have thus far isolated Tc1 insertions in 16 different genes and obtained deletion derivatives of 6 of those.
Amyloid precursor protein (APP) is a member of a larger gene family including amyloid precursor-like proteins (APLP), APLP2 and APLP1. To examine the function of APLP2 in vivo, we generated APLP2 knockout (KO) mice. They are of normal size, fertile, and appear healthy up to 22 months of age. We observed no impaired axonal outgrowth of olfactory sensory neurons following bulbectomy, suggesting against an important role for APLP2 alone in this process. Because APLP2 and APP are highly homologous and may serve similar functions in vivo, we generated mice with targeted APLP2 and APP alleles. Approximately 80% of double KO mice die within the first week after birth, suggesting that APLP2 and APP are required for early postnatal development. The surviving approximately 20% of double KO mice are 20-30% reduced in weight and show difficulty in righting, ataxia, spinning behavior, and a head tilt, suggesting a deficit in balance and/or strength. Adult double KO mice mate poorly, despite apparent normal ovarian and testicular development. Otherwise, double KO mice appear healthy up to 13 months of age. We conclude, that APLP2 and APP can substitute for each other functionally.
The let-60 ras gene acts in a signal transduction pathway to control vulval differentiation in Caenorhabditis elegans. By screening suppressors of a dominant negativelet-60 ras allele, we isolated three loss-of-function mutations in the sur-5 gene which appear to act as negative regulators of let-60 ras during vulval induction.sur-5 mutations do not cause an obvious mutant phenotype of their own, and they appear to specifically suppress only one of the
two groups of let-60 ras dominant negative mutations, suggesting that the gene may be involved in a specific aspect of Ras activation. Consistent
with its negative function, overexpressing sur-5 from an extragenic array partially suppresses the Multivulva phenotype of an activatedlet-60 ras mutation and causes synergistic phenotypes with a lin-45 raf mutation. We have clonedsur-5 and shown that it encodes a novel protein. We have also identified a potential mammalian SUR-5 homolog that is about 35%
identical to the worm protein. SUR-5 also has some sequence similarity to acetyl coenzyme A synthetases and is predicted to
contain ATP/GTP and AMP binding sites. Our results suggest thatsur-5 gene function may be conserved through evolution.
Studies of the development of the nematode Caenorhabditis elegans established that programmed cell death involves specific genes and proteins and that those genes and proteins act within the cells that die. This finding revealed that cell death is a fundamental and active biological process, much like cell division and cell differentiation. The characterization of genes responsible for programmed cell death in C. elegans has defined a molecular genetic pathway. This pathway is conserved evolutionarily and provides a basis for understanding programmed cell death in more complex organisms, including humans. Knowledge of the mechanisms of programmed cell death should help lead to new methods for the diagnosis and treatment of human diseases characterized by too many or too few cell deaths, including cancer.
The amyloid-beta precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated amyloid-beta (A beta) peptide formation. The predominant site of caspase-mediated proteolysis is within the cytoplasmic tail of APP, and cleavage at this site occurs in hippocampal neurons in vivo following acute excitotoxic or ischemic brain injury. Caspase-3 is the predominant caspase involved in APP cleavage, consistent with its marked elevation in dying neurons of Alzheimer's disease brains and colocalization of its APP cleavage product with A beta in senile plaques. Caspases thus appear to play a dual role in proteolytic processing of APP and the resulting propensity for A beta peptide formation, as well as in the ultimate apoptotic death of neurons in Alzheimer's disease.
Amyloid-beta precursor protein (APP), a widely expressed cell-surface protein, is cleaved in the transmembrane region by gamma-secretase. gamma-Cleavage of APP produces the extracellular amyloid beta-peptide of Alzheimer's disease and releases an intracellular tail fragment of unknown physiological function. We now demonstrate that the cytoplasmic tail of APP forms a multimeric complex with the nuclear adaptor protein Fe65 and the histone acetyltransferase Tip60. This complex potently stimulates transcription via heterologous Gal4- or LexA-DNA binding domains, suggesting that release of the cytoplasmic tail of APP by gamma-cleavage may function in gene expression.
In C. elegans, a hyperactivated MEC-4(d) ion channel induces necrotic-like neuronal death that is distinct from apoptosis. We report that null mutations in calreticulin suppress both mec-4(d)-induced cell death and the necrotic cell death induced by expression of a constitutively activated Galpha(S) subunit. RNAi-mediated knockdown of calnexin, mutations in the ER Ca(2+) release channels unc-68 (ryanodine receptor) or itr-1 (inositol 1,4,5 triphosphate receptor), and pharmacological manipulations that block ER Ca(2+) release also suppress death. Conversely, thapsigargin-induced ER Ca(2+) release can restore mec-4(d)-induced cell death when calreticulin is absent. We conclude that high [Ca(2+)](i) is a requirement for necrosis in C. elegans and suggest that an essential step in the death mechanism is release of ER-based Ca(2+) stores. ER-driven Ca(2+) release has previously been implicated in mammalian necrosis, suggesting necrotic death mechanisms may be conserved.
The multigenic family of mammalian Fe65s encodes three highly similar proteins with the same modular organisation: a WW domain and two phosphotyrosine-binding domains. The PTB2 domain of these proteins binds to the cytosolic domains of the Alzheimer's β-amyloid precursor protein APP and related proteins APLP1 and APLP2, generating a highly redundant system that is hard to dissect by reverse genetics. By searching potential Fe65-like genes in the nematode Caenorhabditis elegans, we identified a single gene, feh-1 (Fe65 homolog-1), encoding a protein with a high sequence similarity to mammalian Fe65s. FEH-1 is also functionally related to mammalian orthologues;in fact its PTB2 domain binds to APL-1, the product of the C. elegansorthologue of APP. Staining with specific antibodies show that the neuromuscular structures of the pharynx are the sites in which FEH-1 is present at highest levels. Expression studies with reporters indicate that the feh-1 gene is also expressed by a subset of the worm neurons.
We generated and isolated a deletion allele of feh-1, and the corresponding homozygous mutants arrest as late embryos or as L1 larvae,demonstrating for the first time an essential role for a Fe65-like gene in vivo. The pharynx of homozygous larvae does not contract and the worms cannot feed. Analysis of pharyngeal pumping in heterozygous worms and in feh-1 RNA-interfered worms indicates that dosage of feh-1function affects the rate of pharyngeal contraction in C. elegans. Interference with apl-1 double-stranded RNA showed a similar effect on pharyngeal pumping, suggesting that FEH-1 and APL-1 are involved in the same pathway. The non-redundant system of the nematode will prove useful for studying the basic biology of the Fe65-APP interaction and the molecular events regulated by this evolutionarily conserved system of interacting proteins.
The beta-amyloid precursor protein has been the focus of much attention from the Alzheimer's disease community for the past decade and a half. The beta-amyloid precursor protein holds a pivotal position in Alzheimer's disease research because it is the precursor to the amyloid beta-protein which many believe plays a central role in Alzheimer's disease pathogenesis. It was also the first gene in which mutations associated with inherited Alzheimer's disease were found. Although the molecular details of the generation of amyloid beta-protein from beta-amyloid precursor protein are being unraveled, the actual physiological functions of beta-amyloid precursor protein are far from clear. This situation is changing as accumulating new evidence suggests that the C-terminal cytosolic tail of beta-amyloid precursor protein may have multiple biological activities, ranging from axonal transport to nuclear signaling. This article reviews the current state of knowledge about the biological functions of beta-amyloid precursor protein.
The human brain contains 100 billion neurons and probably one thousand times more synapses. Such a system can be analyzed at different complexity levels, from cognitive functions to molecular structure of ion channels. However, it remains extremely difficult to establish links between these different levels. An alternative strategy relies on the use of much simpler animals that can be easily manipulated. In 1974, S. Brenner introduced the nematode Caenorhabditis elegans as a model system. This worm has a simple nervous system that only contains 302 neurons and about 7,000 synapses. Forward genetic screens are powerful tools to identify genes required for specific neuron functions and behaviors. Moreover, studies of mutant phenotypes can identify the function of a protein in the nervous system. The data that have been obtained in C. elegans demonstrate a fascinating conservation of the molecular and cellular biology of the neuron between worms and mammals through more than 550 million years of evolution.
Neurons require long-distance microtubule-based transport systems to ferry vital cellular cargoes and signals between cell bodies and axonal or dendritic terminals. Considerable progress has been made on developing a molecular understanding of these processes and how they are integrated into normal neuronal functions. Recent work also suggests that these transport systems may fail early in the pathogenesis of a number of neurodegenerative diseases.
gamma-Secretase catalyzes intramembrane proteolysis of various type I membrane proteins, including the amyloid-beta precursor protein and the Notch receptor. Despite its importance in the pathogenesis of Alzheimer's disease and to normal development, this protease has eluded identification until only very recently. Four membrane proteins are now known to be members of the protease complex: presenilin, nicastrin, aph-1, and pen-2. Recent findings suggest that these four proteins are sufficient to reconstitute the active gamma-secretase complex and that together they mediate the cell surface signaling of a variety of receptors via intramembrane proteolysis.
Alzheimer's disease (AD) is a neurodegenerative disease associated with progressive dementia. This mini-review focuses on how the amyloid precursor protein (APP) plays a central role in AD and Down syndrome as the regulator of the APP-BP1/hUba3 activated neddylation pathway. It is argued that the physiological function of APP is to downregulate the level of beta-catenin. However, this APP function is abnormally amplified in patients with familial AD (FAD) mutations in APP and presenilins, resulting in the hyperactivation of neddylation and the decrease of beta-catenin below a threshold level. Evidence in the literature is summarized to show that dysfunction of APP in downregulating beta-catenin may underlie the mechanism of neuronal death in AD and Down syndrome.
Cleavage of amyloid-beta precursor protein (APP) by site-specific proteases generates amyloid-beta peptides (Abetas), which are thought to induce Alzheimer's disease. We have identified an independently folded extracellular domain of human APP localized proximal to the Abeta sequence, and determined the three-dimensional structure of this domain by NMR spectroscopy. The domain is composed of four alpha-helices, three of which form a tight antiparallel bundle, and constitutes the C-terminal half of the central extracellular region of APP that has been implicated in the regulation of APP cleavage. Sequence comparisons demonstrate that the domain is highly conserved among all members of the APP family, including invertebrate homologues, suggesting an important role for this region in the biological function of APP. The identification of this domain and the availability of its atomic structure will facilitate analysis of APP function and of the role of the extracellular region in the regulation of APP cleavage.
Amyloid beta-peptide, which forms neuronal and vascular amyloid deposits in Alzheimer's disease, is derived from an integral membrane protein precursor. The biological function of the precursor is currently unclear. Here we describe the X-ray structure of E2, the largest of the three conserved domains of the precursor. The structure of E2 consists of two coiled-coil substructures connected through a continuous helix and bears an unexpected resemblance to the spectrin family of protein structures. E2 can reversibly dimerize in the solution, and the dimerization occurs along the longest dimension of the molecule in an antiparallel orientation, which enables the N-terminal substructure of one monomer to pack against the C-terminal substructure of a second monomer. Heparan sulfate proteoglycans, the putative ligand for the precursor present in extracellular matrix, bind to E2 at a conserved and positively charged site near the dimer interface.
The amyloid precursor protein (APP) must fulfill important roles based on its sequence conservation from fly to human. Although multiple functions for APP have been proposed, the best-known role for this protein is as the precursor of Abeta peptide, a neurotoxic 39-43-amino acid peptide crucial to the pathogenesis of Alzheimer's disease. To investigate additional roles for APP with an eye toward understanding the molecular basis of the pleiotropic effects ascribed to APP, we isolated proteins that interacted with the plasma membrane isoform of APP. We employed a membrane-impermeable crosslinker to immobilize proteins binding to transmembrane APP in human embryonic kidney (HEK)293 cells expressing APP751 (HEK275) or rat embryonic day 18 primary neurons infected with a virus expressing APP. Notch2 was identified as a potential APP binding partner based on mass spectrometry analysis of APP complexes immunopurified from neurons. To confirm the interaction between Notch2 and APP, we carried out immunoprecipitation studies in HEK275 cells transiently expressing full-length Notch2 using Notch2 antibodies. The results indicated that APP and Notch2 interact in mammalian cells, and confirmed our initial findings. Interestingly, Notch1 also coimmunoprecipitated with APP, suggesting that APP and Notch family members may engage in intermolecular cross talk to modulate cell function. Finally, cotransfection of APP/CFP and Notch2/YFP into COS cells revealed that these two proteins colocalize on the plasma membrane. Intracellularly, however, although some APP and Notch molecules colocalize, others reside in distinct locations. The discovery of proteins that interact with APP may aid in the identification of new functions for APP.
- Gg Glenner
- Cw Wong
Glenner GG, Wong CW (1984) Biochem Biophys Res Commun 120:885–890.
- C Reinhard
- Ss Hébert
Reinhard C, Hébert SS, De Strooper B (2005) EMBO J 24:3996–4006.