scholarly journals Modeling neurodegeneration in Caenorhabditiselegans

2020 ◽  
Vol 13 (10) ◽  
pp. dmm046110
Author(s):  
Kim A. Caldwell ◽  
Corey W. Willicott ◽  
Guy A. Caldwell
Keyword(s):  
Neurodegenerative Diseases ◽  
Drug Targets ◽  
Fluorescent Proteins ◽  
Critical Threshold ◽  
C Elegans ◽  
Chemical Genetic ◽  
Modifying Factors ◽  
Genes Encoding ◽  
The Impact

ABSTRACTThe 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.

scholarly journals Quantitative assessment of near-infrared fluorescent proteins

2021 ◽  
Author(s):  
Kiryl Piatkevich ◽  
Hanbin Zhang ◽  
Stavrini Papadaki ◽  
Xiaoting Sun ◽  
Luxia Yao ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Quantitative Assessment ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Oligomeric State ◽  
C Elegans ◽  
The Right ◽  
Infrared Fluorescent Protein

Abstract Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins, which are rapidly becoming popular probes for a variety of imaging applications. To assist end-users with a selection of the right near-infrared fluorescent protein for a given application, we will conduct a quantitative assessment of intracellular brightness, photostability, and oligomeric state of 19 near-infrared fluorescent proteins in cultured mammalian cells. The top-performing proteins will be further validated for in vivo imaging of neurons in C. elegans, zebrafish, and mice. We will also assess the applicability of the selected NIR FPs for expansion microscopy and two-photon imaging.

scholarly journals Caenorhabditis elegans model for admixed proteinopathy of Huntington’s and Alzheimer-type

2019 ◽  
Author(s):  
Wadim J. Kapulkin
Keyword(s):  
Caenorhabditis Elegans ◽  
Clinical Symptoms ◽  
Fluorescent Proteins ◽  
Amyloid Formation ◽  
Dependent Manner ◽  
Huntington's Chorea ◽  
C Elegans ◽  
Pathogenic Variants ◽  
Models Of Disease

ABSTRACTFormation of proteinaceous deposits composed of abnormally aggregated proteins characterizes a range of pathological conditions. Proteinaceous inclusions detected in the neurodegenerative conditions such Huntington’s chorea (HD) and Alzheimer’s disease (AD) are often referred as pathognomic and regarded as causally implicated. Despite of differences in aetiology and underlying genetics, rare cases of combined HD and AD were reported and described as admixed proteinopathies. Mixed proteopathies are characterized by the co-occurrence of at least two types of abnormal aggregation-prone variants; pathological deposition of proteinaceous inclusions might however, affect different cell populations. Here, combining plaques derived from human ß-amyloid with mutant HTT-like polyglutamine inclusions in a cell-autonomous manner, we report on the Caenorhabditis elegans model for admixed proteinopathy of Alzheimer’s and Huntington’s type. We show both types of intracellular foci are formed in vivo: non-amyloidic extended polyglutamine derived inclusions and distinguish those from the presence of mature ß- amyloid fibers. We found that polyglutamines expanded above pathogenic threshold and ß-amyloid act synergistically to promote the progression of proteotoxicity in a temperature dependent manner. We further, implicate the hsp-1 (the predominant C. elegans chaperone interacting with ER-routed Aß42) modulate the proteotoxic insults observed in combined proteopathy model. Our results demonstrate how the in vivo model of admixed proteopathy could be utilized to probe for human pathogenic variants confined to the same cellular type. In that perspective expanded aggregation-prone polyglutamines appended with fluorescent proteins could be regarded as ‘pathogenic probes’ useful in the proteotoxicity assays i.e. involving ß-amyloid and possibly other comparable models of disease-associated aggregation prone variants.We anticipate models of combined proteopathies will be informative regarding the underlying pathogenesis and provide the sensitized background for sophisticated screening. For example the combinatorial effects of multiple pathogenic aggregation-prone variants could be tested against mutant backgrounds and pharmacological compounds. Furthermore, we surmise the postulated synergistic actions might explain some of the overlaps in observed progression of clinical symptoms in HD and AD. We also formulate the conjecture regarding the polyQ containing proteins as a contributing factor in degenerative conditions associated with increased ß-amyloid formation and deposition.

Fluorescent Nanodiamond – A Novel Nanomaterial for In Vivo Applications

2011 ◽  
Vol 1362 ◽  
Author(s):  
Nitin Mohan ◽  
Bailin Zhang ◽  
Cheng-Chun Chang ◽  
Liling Yang ◽  
Chao-Sheng Chen ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Ion Irradiation ◽  
Spectroscopic Characterization ◽  
Correlation Spectroscopy ◽  
Model Organisms ◽  
C Elegans ◽  
Red Fluorescent Proteins ◽  
Fluorescent Nanodiamonds ◽  
High Fluorescence

ABSTRACTFluorescent nanodiamonds (FNDs) with a size in the range of 10 – 100 nm have been produced by ion irradiation and annealing, and isolated by differential centrifugation. Single particle spectroscopic characterization with confocal fluorescence microscopy and fluorescence correlation spectroscopy indicates that they are photostable and useful as an alternative to far-red fluorescent proteins for bioimaging applications. We demonstrate the application by performing in vivo imaging of bare and bioconjugated FND particles (100 nm in diameter) in C. elegans and zebrafishes and exploring the interactions between this novel nanomaterial and the model organisms. Our results indicate that FNDs can be delivered to the embryos of both organisms by microinjection and eventually into the hatched larvae in the next generation. No deleterious effects have been observed for the carbon-based nanoparticles in vivo. The high fluorescence brightness, excellent photostability, and nontoxic nature of the nanomaterial have allowed long-term imaging and tracking of embryogenesis in the organisms.

scholarly journals Complex Regulation Pathways of AmpC-Mediated β-Lactam Resistance in Enterobacter cloacae Complex

2015 ◽  
Vol 59 (12) ◽  
pp. 7753-7761 ◽  
Author(s):  
François Guérin ◽  
Christophe Isnard ◽  
Vincent Cattoir ◽  
Jean Christophe Giard
Keyword(s):  
Drug Targets ◽  
Genetic Regulation ◽  
Enterobacter Cloacae ◽  
Opportunistic Pathogen ◽  
Regulation Mechanism ◽  
Content Type ◽  
Genes Encoding ◽  
High Concentrations ◽  
High Level

ABSTRACTEnterobacter cloacaecomplex (ECC), an opportunistic pathogen causing numerous infections in hospitalized patients worldwide, is able to resist β-lactams mainly by producing the AmpC β-lactamase enzyme. AmpC expression is highly inducible in the presence of some β-lactams, but the underlying genetic regulation, which is intricately linked to peptidoglycan recycling, is still poorly understood. In this study, we constructed different mutant strains that were affected in genes encoding enzymes suspected to be involved in this pathway. As expected, the inactivation ofampC,ampR(which encodes the regulator protein ofampC), andampG(encoding a permease) abolished β-lactam resistance. Reverse transcription-quantitative PCR (qRT-PCR) experiments combined with phenotypic studies showed that cefotaxime (at high concentrations) and cefoxitin induced the expression ofampCin different ways: one involving NagZ (aN-acetyl-β-d-glucosaminidase) and another independent of NagZ. Unlike the model established forPseudomonas aeruginosa, inactivation of DacB (also known as PBP4) was not responsible for a constitutiveampCoverexpression in ECC, whereas it caused AmpC-mediated high-level β-lactam resistance, suggesting a post-transcriptional regulation mechanism. Global transcriptomic analysis by transcriptome sequencing (RNA-seq) of adacBdeletion mutant confirmed these results. Lastly, analysis of 37 ECC clinical isolates showed that amino acid changes in the AmpD sequence were likely the most crucial event involved in the development of high-level β-lactam resistancein vivoas opposed toP. aeruginosawheredacBmutations have been commonly found. These findings bring new elements for a better understanding of β-lactam resistance in ECC, which is essential for the identification of novel potential drug targets.

scholarly journals Characterization of fluorescent proteins with intramolecular photostabilization

2020 ◽  
Author(s):  
Sarah S. Henrikus ◽  
Konstantinos Tassis ◽  
Lei Zhang ◽  
Jasper H. M. van der Velde ◽  
Christian Gebhardt ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Photophysical Properties ◽  
Unnatural Amino Acid ◽  
Biological Research ◽  
Triplet Energy ◽  
Organic Fluorophores ◽  
Fluorescent Tags ◽  
The Impact

AbstractGenetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Since there are no other natural fluorescent tags with comparable features, the impact of fluorescent proteins for biological research cannot be overemphasized. Despite their importance, their photophysical properties, i.e., brightness, count-rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties. The approach uses direct conjugation of photostablizing compounds such as triplet-state quenchers or redox-active substances to an organic fluorophore, thereby creating high local concentrations of photostabilizer. Here, we introduce an experimental strategy to screen for the effects of covalently-linked photostabilizers on fluorescent proteins. We recombinantly produced a double cysteine mutant (A206C/L221C) of α-GFP for attachment of photostabilizer-maleimides on the ß-barrel in close proximity to the chromophore. Whereas labelling with photostabilizers such as Trolox, Nitrophenyl, and Cyclooctatetraene, which are often used for organic fluorophores, had no effect on α-GFP-photostability, a substantial increase of photostability was found upon conjugation of α-GFP to an azobenzene derivative. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet-energy of azobenzene might be crucial for triplet-quenching of fluorophores in the near-UV and blue spectral range. Our study paves the way towards the development and design of a second generation of fluorescent proteins with photostabilizers placed directly in the protein barrel by methods such as unnatural amino acid incorporation.

scholarly journals ATP13A2-mediated endo-lysosomal polyamine export counters mitochondrial oxidative stress

2020 ◽  
Vol 117 (49) ◽  
pp. 31198-31207
Author(s):  
Stephanie Vrijsen ◽  
Laura Besora-Casals ◽  
Sarah van Veen ◽  
Jeffrey Zielich ◽  
Chris Van den Haute ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Antioxidant Properties ◽  
Loss Of Function ◽  
Mitochondrial Oxidative Stress ◽  
C Elegans ◽  
Polyamine Transport ◽  
Exogenous Spermine ◽  
The Impact

Recessive loss-of-function mutations inATP13A2(PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson’s disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in aCaenorhabditis elegansstrain deficient in the ATP13A2 orthologcatp-6. These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated byatfs-1, theC. elegansortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.

scholarly journals Impact of Bicarbonate on PBP2a Production, Maturation, and Functionality in Methicillin-Resistant Staphylococcus aureus (MRSA)

2021 ◽  
Author(s):  
Selvi C. Ersoy ◽  
Henry F. Chambers ◽  
Richard A. Proctor ◽  
Adriana E. Rosato ◽  
Nagendra N. Mishra ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Ex Vivo ◽  
Carotenoid Content ◽  
Methicillin Resistant ◽  
Genes Encoding ◽  
Regulatory Impact ◽  
Mrsa Strains ◽  
The Impact

Certain methicillin-resistant Staphylococcus aureus (MRSA) strains exhibit β-lactam-susceptibility in vitro, ex vivo and in vivo in the presence of NaHCO3 (NaHCO3-responsive MRSA). Herein, we investigate the impact of NaHCO3 on factors required for PBP2a functionality. Prototype NaHCO3-responsive and -nonresponsive MRSA strains (as defined in vitro) were assessed for the impact of NaHCO3 on: expression of genes involved in PBP2a production-maturation pathways (mecA, blaZ, pbp4, vraSR, prsA, sigB, and floA); membrane PBP2a and PrsA protein content; and membrane carotenoid content. Following NaHCO3 exposure in NaHCO3-responsive (vs - nonresponsive) MRSA, there was significantly reduced expression of: i) mecA and blaZ; ii) the vraSR-prsA gene axis; and iii) pbp4. Carotenoid production was reduced, while floA expression was increased by NaHCO3 exposure in all MRSA strains. This work underscores the distinct regulatory impact of NaHCO3 on a cadre of genes encoding factors required for maintenance of the MRSA phenotype through PBP2a functionality and maturation.

scholarly journals Extracellular matrix regulates morphogenesis and function of ciliated sensory organs in Caenorhabditis elegans

2018 ◽  
Author(s):  
Deanna M. De Vore ◽  
Karla M. Knobel ◽  
Ken C.Q. Nguyen ◽  
David H. Hall ◽  
Maureen M. Barr
Keyword(s):  
Extracellular Matrix ◽  
Dendritic Morphology ◽  
C Elegans ◽  
Genes Encoding ◽  
Health And Disease ◽  
Autosomal Dominant Polycystic Kidney ◽  
Signaling Organelles ◽  
And Function

ABSTRACTCilia and extracellular vesicles (EVs) are signaling organelles that play important roles in human health and disease. In C. elegans and mammals, the Autosomal Dominant Polycystic Kidney Disease (ADPKD) gene products polycystin-1 and polycystin-2 localize to both cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation. Hence, the nematode offers an excellent system in which to address central questions regarding the biology of cilia, EVs, and the polycystins. We discovered an unexpected role of the mec-1, mec-5, and mec-9 genes encoding extracellular matrix (ECM) components. We determined that these ECM encoding genes regulate polycystin localization and function, ciliary EV release, cilia length, dendritic morphology, and neuron-glia interactions. Abnormal ECM and fibrosis are observed in ciliopathies such as ADPKD, nephronophthisis, and Bardet-Biedl Syndrome. Our studies reveal multifaceted roles for ECM proteins in the ciliated nervous system of the worm and provide a powerful new in vivo model to study the relationship between ECM, the polycystins, and ciliopathies.

scholarly journals Therapeutics Effects of Oral Cavity Derived Stem Cells on the Neurodegenerative Diseases: A Systematic Review

2021 ◽  
pp. 1-29
Author(s):  
Emel Uzunoglu-Ozyurek ◽  
◽  
Gizem Önal ◽  
Serap Dökmeci ◽  
◽  
...  
Keyword(s):  
Stem Cells ◽  
Oral Cavity ◽  
Neurodegenerative Diseases ◽  
In Vitro Studies ◽  
In Vivo Studies ◽  
Published Data ◽  
Moderate Evidence ◽  
The Impact

Objective: Published data obtained from in vitro and in vivo studies was reviewed systematically and analyzed critically in order to evaluate the effect of oral cavity derived stem cells (OCDSCs) on the recovery/therapy of neurodegenerative diseases (NDs) such as Alzheimer’s (AD), Amyotrophic Lateral Sclerosis (ALS), Huntington’s (HD) and Parkinson’s (PD). Methods: An electronic search was accomplished. References of included articles were also searched manually. Studies were critically evaluated for suitability against inclusion/exclusion criteria and data was extracted. Bias risk evaluation of the studies and evidence synthesis were conducted. Results: Fourteen in vivo and 10 in vitro studies met inclusion criteria. PD was induced in 10 in vivo and 7 in vitro studies, while AD was induced in 2 in vivo and 4 in vitro studies. Two studies (1 in vitro and 1 in vivo) evaluated ALS disease and 1 in vivo study evaluated HD. Moderate evidence was found for in vitro studies reporting positive effect of OCDSCs on either PD or AD recovery. Strong evidence was found for in vivo studies in which PD animal models were used; while moderate evidence was found for the impact of OCDSCs on the recovery of the AD. Limited evidence was found for in vivo studies evaluating HD and ALS. Conclusion: Although studies included reported favorable data regarding the OCDSCs on NDs, they presented considerable risk of bias. Because of heterogenous study characteristics, current study recommends the improvement of standardized methods to evaluate the therapeutics effects of OCDSCs on the NDs.

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