Biology subjects

Ackley, B. D.

Publications and source records attributed to Ackley, B. D..

8 recordsLinked to original sources

bioRxiv2026

Tropism of Drosophila innubila Nudivirus in Cell Culture and in Tissues

Most viruses are successful because they can infect and exploit specific host tissues for reproduction and transmission. As a result, we often classify viruses by their ability to infect different tissues: respiratory, reproductive and gut, for example. Thus, knowing the tissue and cellular tropism of a virus may elucidate aspects of its biology including routes of transmission and virulence. We studied tropism of the Drosophila innubila Nudivirus (DiNV) in cells and infected hosts. DiNV is a nudivirus, a family of viruses related to the baculoviruses that are relatively large (150-200 nm), contain large double-stranded DNA genomes, and infect different host tissues. Cultured cells stained with an antibody against the DiNV nucleocapsid demonstrated punctate staining in the cytoplasm by confocal microscopy. Electron micrographs of infected cells, on the other hand demonstrated that virions are present in the nucleus. After purification, DiNV was often associated with multimembrane-like vesicles as described for Oryctes rhinoceros nudivirus. We examined tropism in hosts first by sagittal section and found most virus associated with the gut and, to a lesser extent, reproductive tissues. Upon dissection and electron microscopy, we found that DiNV in the gut is primarily in the visceral muscle of the gut and not the lumen as might be expected for a virus if transmitted fecal-orally.

microbiology↗

bioRxiv2025

A C. elegans model of familial Alzheimer's disease shows age-dependent synaptic degeneration independent of amyloid β-peptide

The membrane-embedded {gamma}-secretase complex is involved in the intramembrane cleavage of [~] 150 substrates. Cleavage of amyloid precursor protein (APP)-derived substrate C99 generates 38-43-residue secreted amyloid {beta}-peptides (A{beta}), with the aggregation-prone 42-residue form (A{beta}42) particularly implicated in the pathogenesis of Alzheimers Disease (AD). However, whether A{beta}42 is the primary driver of neurodegeneration in AD remains unclear. Dominant mutations in APP or presenilin--the catalytic component of {gamma}-secretase--cause early-onset familial AD (FAD) and reduce one or more steps in the multi-step processive proteolysis of C99 to A{beta} peptides, apparently through stabilization of {gamma}-secretase enzyme-substrate (E-S) complexes. To investigate mechanisms of neurodegeneration in FAD, we developed new C. elegans models co-expressing wild-type or FAD-mutant C99 substrate and presenilin-1 (PSEN1) variants in neurons, allowing intramembrane processing of C99 to A{beta} in vivo. We demonstrate that while FAD-mutation of either C99 or PSEN1 leads to age-dependent synaptic loss, proteolytically inactive PSEN1 did not. Designed mutations that allow stable E-S complex formation without A{beta}42 or A{beta} production likewise result in synaptic degeneration. Moreover, replacement of C99 with variants of a Notch1-based substrate revealed that disrupted processing of another {gamma}-secretase substrate can similarly lead to synaptic degeneration. These results support a model in which synaptic loss can be triggered by toxic, stalled {gamma}-secretase E-S complexes in the absence of A{beta} production and not by simple loss of proteolytic function. This new C. elegans system provides a powerful platform to study the role of dysfunctional {gamma}-secretase substrate processing in FAD pathogenesis. SIGNIFICANCEDominantly inherited familial Alzheimers disease (FAD) is caused by mutations in the enzyme or substrate that produces amyloid {beta}-peptides (A{beta}). These mutations alter enzyme processing of substrate to A{beta} and can skew production of A{beta} to aggregation-prone forms that deposit as pathological plaques. Nevertheless, whether A{beta} is the primary driver of AD remains unresolved. Recent evidence supports a model in which loss of neuronal connections in FAD is due to the stalled enzymatic process of A{beta} production, rather than the A{beta} products. Using the roundworm C. elegans as a genetic model system, we show here that the stalled process itself, rather than A{beta} or reduced enzyme activity, can trigger loss of neuronal connections with age in this simple model animal.

neuroscience↗

bioRxiv2023

Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes

Alzheimers disease is characterized pathologically by cerebral deposition of 42-residue amyloid {beta}-peptide (A{beta}42), proteolytically produced from amyloid precursor protein (APP) by {beta}- and {gamma}-secretases.1 Although mutations in APP and presenilin, the catalytic component of {gamma}-secretase, cause familial Alzheimers disease (FAD), a role for A{beta}42 as the primary disease driver has not been clearly established and remains controversial.2,3 Here we show through comprehensive analysis of the multi-step proteolysis of APP substrate C99 by {gamma}-secretase that FAD mutations are consistently deficient in early proteolytic events, not later events that produce secreted A{beta} peptides. Cryo-electron microscopy revealed that a substrate mimetic traps {gamma}-secretase at the transition state for intramembrane proteolysis, and this structure closely aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and fluorescence lifetime imaging microscopy in cultured cells support stabilization by FAD mutations of enzyme-substrate and/or enzyme-intermediate complexes. Neuronal expression of C99 and/or presenilin-1 in Caenorabditis elegans led to age-dependent synaptic loss only when one of the transgenes carried an FAD mutation. Designed mutations that stabilize the enzyme-substrate complex and block proteolysis likewise led to synaptic loss. Collectively, these findings implicate the stalled process--not the released products--of {gamma}-secretase cleavage of substrates in FAD pathogenesis.

neuroscience↗

bioRxiv2021

Distinct roles for two Caenorhabditis elegans acid-sensing ion channels in an ultradian clock

Biological clocks are fundamental to an organisms health, controlling periodicity of behavior and metabolism. Here, we identify two acid-sensing ion channels, with very different proton sensing properties, and describe their role in an ultradian clock, the defecation motor program (DMP) of the nematode Caenorhabditis elegans. An ACD-5-containing channel, on the apical membrane of the intestinal epithelium, is essential for maintenance of luminal acidity, and thus the rhythmic oscillations in lumen pH. In contrast, the second channel, composed of FLR-1, ACD-3 and/or DEL-5, located on the basolateral membrane, controls the intracellular Ca2+ wave and forms a core component of the master oscillator that controls timing and rhythmicity of the DMP. flr-1 and acd-3/del-5 mutants show severe developmental and metabolic defects. We thus directly link the proton-sensing properties of these channels to their physiological roles in pH regulation and Ca2+ signaling, the generation of an ultradian oscillator, and its metabolic consequences. One-Sentence SummaryTwo acid-sensing DEG/ENaC channels play distinct roles in controlling different aspects of rhythmic proton and Ca2+ oscillations in the C. elegans intestine.

physiology↗

bioRxiv2021

Wild-type Caenorhabditis elegans Isolates Exhibit Distinct Gene Expression Profiles in Response to Microbial Infection

The soil-dwelling nematode Caenorhabditis elegans serves as a model system to study innate immunity against microbial pathogens. C. elegans have been collected from around the world, where they, presumably, adapted to regional microbial ecologies. Here we use survival assays and RNA- sequencing to better understand how two isolates from disparate climates respond to pathogenic bacteria. We found that, relative to N2 (originally isolated in Bristol, UK), CB4856 (isolated in Hawaii), was more susceptible to the Gram-positive pathogen, Staphylococcus epidermidis, but equally susceptible to Staphylococcus aureus as well as two Gram-negative pathogens, Providencia rettgeri and Pseudomonas aeruginosa. We performed transcriptome analysis of infected worms and found gene- expression profiles were considerably different in an isolate-specific and pathogen-specific manner. We performed GO term analysis to categorize differential gene expression in response to S. epidermidis. In N2, genes that encoded detoxification enzymes and extracellular matrix proteins were significantly enriched, while in CB4856, genes that encoded detoxification enzymes, C-type lectins, and lipid metabolism proteins were enriched, suggesting they have different responses to these bacterial pathogens, despite being the same species. Overall, discerning gene expression signatures in an isolate by pathogen manner can help us to understand the different possibilities for the evolution of immune responses within organisms.

genomics↗

bioRxiv2020

A comparative analysis of Caenorhabditis and Drosophila transcriptional changes in response to pathogen infection

Drosophila melanogaster and Caenorhabditis elegans are well-used invertebrate models for studying the innate immune system. The organisms are susceptible to bacterial pathogens that include Pseudomonas species, (entomophilia - Drosophila) or (aeruginosa - Caenorhabditis), E. faecalis and P. rettgeri, which are or are related to human pathogens. Further, the consequences of exposure to these pathogens, in terms of organismal survival, are roughly equivalent when compared. That is, worms and flies are more susceptible to infection by Pseudomonas than E. faecalis, whereas organismal survival on E. faecalis and P. rettgeri are roughly the same in both. To better understand how these organisms are coordinating their responses to these bacterial pathogens we examined transcriptomes in infected animals. We grouped our analysis based on protein orthology. Of the 3611 pairs analyzed, we found genes whose responses were conserved across the different species at a higher than expected rate for two of the three pathogens. Interestingly within the animals, genes with 1:1 orthologs between species behaved differently. Such genes were more likely to be expressed in D. melanogaster, and less likely to be expressed in C. elegans. From this analysis we found that the gene nucleobindin (nucb-1/NUCB1 in C. elegans and D. melanogaster, respectively) was upregulated in both species in response to Gram negative bacteria. We used RNAi to knock down nucb-1 and found the treated animals were more susceptible to infection by the Gram negative pathogen P. rettgeri than controls. These results provide insight into some of the conserved mechanisms of pathogen defense, but also suggest that these divergent organisms have evolved specific means to orchestrate the defense against pathogens. Article SummaryWe analyzed transcriptomic data from C. elegans and D. melanogaster to compare the expression of orthologous pairs of genes in response to bacterial pathogens. Our results indicated that only a handful of genes that are orthologous between species are differentially expressed in response to pathogens, but that the pattern of expression was different when comparing one-to-one orthologs versus those that are restricted to one of the two organisms. These results suggest that, although broad patterns of susceptibility to bacterial pathogens are conserved, the regulatory framework by which the organisms fight pathogens is less well conserved. Further our results suggest a more complete analysis of the evolutionary changes in organismal responses to pathogens is required.

genetics↗

bioRxiv2018

The C. elegans CHP1 homolog, pbo-1, functions in innate immunity by regulating the pH of the intestinal lumen

Caenorhabditis elegans are soil-dwelling nematodes and models for understanding innate immunity and infection. Previous work has described a regularly-timed pH change in the intestine of Caenorhabditis elegans called the pH wave. To characterize this wave and its function in the worm, we developed a novel fluorescent dye (KR35) that accumulates in the intestine and sensitively responds to dynamic changes in pH. Here, we use KR35 to show that mutations in the Ca2+-binding protein, PBO-1 abrogate the pH wave, causing the anterior intestine to be constantly acidic. Surprisingly, pbo-1 mutants were also more susceptible to infection by several bacterial pathogens. We could suppress pathogen susceptibility in pbo-1 mutants by treating the animals with pH-buffering bicarbonate, suggesting the pathogen susceptibility is a function of the acidity of the intestinal pH. Furthermore, we use KR35 to show that pathogens completely neutralize the pH in the intestine of wild type, but not pbo-1 mutants. C. elegans is known to increase production of reactive oxygen species (ROS), such as H2O2, in response to pathogens, which is an important component of pathogen defense. We show that pbo-1 mutants exhibited decreased H2O2 in response to pathogens, which could also be partially restored in pbo-1 animals treated with bicarbonate. Ultimately, our results support a model whereby pbo-1 functions during infection to permit pH changes in the intestine that are important for fighting pathogens.\n\nAuthor SummaryInnate immunity is critical for host defense against pathogens. However, questions remain about how the host senses and responds to pathogen invasion. Using a pH-sensitive fluorescent dye and a Caenorhabditis elegans pathogen infection model we show that pathogens induce changes in pH of the worm intestine. We also show that intestinal pH directly affects production of reactive oxygen species (e.g. H2O2) important for pathogen defense. Our results show that pH regulation is an important component of the innate immune response to pathogens.

microbiology↗

bioRxiv2018

Intermediate filaments EXC-2 and IFA-4 Maintain Luminal Structure of the Tubular Excretory Canals in Caenorhabditis elegans

The excretory canals of Caenorhabditis elegans are a model for understanding the maintenance of apical morphology in narrow single-celled tubes. Light and electron microscopy shows that mutants in exc-2 start to form canals normally, but these swell to develop large fluid-filled cysts that lack a complete terminal web at the apical surface, and accumulate filamentous material in the canal lumen. Here, whole-genome sequencing and gene rescue show that exc-2 encodes intermediate filament protein IFC-2. EXC-2/IFC-2 protein, fluorescently tagged via CRISPR/Cas9, is located at the apical surface of the canals independently of other intermediate filament proteins. EXC-2 is also located in several other tissues, though the tagged isoforms are not seen in the larger intestinal tube. Tagged EXC-2 binds via pulldown to intermediate filament protein IFA-4, which is also shown to line the canal apical surface. Overexpression of either protein results in narrow but shortened canals. These results are consistent with a model whereby three intermediate filaments in the canals, EXC-2, IFA-4, and IFB-1, restrain swelling of narrow tubules in concert with actin filaments that guide the extension and direction of tubule outgrowth, while allowing the tube to bend as the animal moves.\n\nArticle SummaryThe C. elegans excretory canals form a useful model for understanding formation of narrow tubes. exc-2 mutants start to form normal canals that then swell into fluid-filled cysts. We show that exc-2 encodes a large intermediate filament (IF) protein previously not thought to be located in the canals. EXC-2 is located at the apical (luminal) membrane, binds to another IF protein, and appears to be one of three IF proteins that form a flexible meshwork to maintain the thin canal diameter. This work provides a genetically useful model for understanding the interactions of IF proteins with other cytoskeletal elements to regulate tube size and growth.

developmental biology↗