Transcriptional signature of prion-induced neurotoxicity in a Drosophila model of transmissible mammalian prion disease

Prion diseases are fatal transmissible neurodegenerative conditions of humans and animals that arise through neurotoxicity induced by PrP misfolding. The cellular and molecular mechanisms of prion-induced neurotoxicity remain undefined. Understanding these processes will underpin therapeutic and control strategies for human and animal prion diseases, respectively. Prion diseases are difficult to study in their natural hosts and require the use of tractable animal models. Here we used RNA-Seq-based transcriptome analysis of prion-exposed Drosophila to probe the mechanism of prion-induced neurotoxicity. Adult Drosophila transgenic for pan neuronal expression of ovine PrP targeted to the plasma membrane exhibit a neurotoxic phenotype evidenced by decreased locomotor activity after exposure to ovine prions at the larval stage. Pathway analysis and quantitative PCR of genes differentially expressed in prion-infected Drosophila revealed up-regulation of cell cycle activity and DNA damage response, followed by down-regulation of eIF2 and mTOR signalling. Mitochondrial dysfunction was identified as the principal toxicity pathway in prion-exposed PrP transgenic Drosophila. The transcriptomic changes we observed were specific to PrP targeted to the plasma membrane since these prion-induced gene expression changes were not evident in similarly treated Drosophila transgenic for cytosolic pan neuronal PrP expression, or in non-transgenic control flies. Collectively, our data indicate that aberrant cell cycle activity, repression of protein synthesis and altered mitochondrial function are key events involved in prion-induced neurotoxicity, and correlate with those identified in mammalian hosts undergoing prion disease. These studies highlight the use of PrP transgenic Drosophila as a genetically well-defined tractable host to study mammalian prion biology.

Download Full-text

Genetic human prion disease modelled in PrP transgenic Drosophila

Biochemical Journal ◽

10.1042/bcj20170462 ◽

2017 ◽

Vol 474 (19) ◽

pp. 3253-3267 ◽

Cited By ~ 3

Author(s):

Alana M. Thackray ◽

Alzbeta Cardova ◽

Hanna Wolf ◽

Lydia Pradl ◽

Ina Vorberg ◽

...

Keyword(s):

Prion Protein ◽

Prion Disease ◽

Prion Diseases ◽

Principal Component ◽

Site Directed Mutagenesis ◽

Host Protein ◽

Therapeutic Interventions ◽

Proteinase K ◽

Human Prion Disease ◽

Transgenic Drosophila

Inherited human prion diseases, such as fatal familial insomnia (FFI) and familial Creutzfeldt–Jakob disease (fCJD), are associated with autosomal dominant mutations in the human prion protein gene PRNP and accumulation of PrPSc, an abnormal isomer of the normal host protein PrPC, in the brain of affected individuals. PrPSc is the principal component of the transmissible neurotoxic prion agent. It is important to identify molecular pathways and cellular processes that regulate prion formation and prion-induced neurotoxicity. This will allow identification of possible therapeutic interventions for individuals with, or at risk from, genetic human prion disease. Increasingly, Drosophila has been used to model human neurodegenerative disease. An important unanswered question is whether genetic prion disease with concomitant spontaneous prion formation can be modelled in Drosophila. We have used pUAST/PhiC31-mediated site-directed mutagenesis to generate Drosophila transgenic for murine or hamster PrP (prion protein) that carry single-codon mutations associated with genetic human prion disease. Mouse or hamster PrP harbouring an FFI (D178N) or fCJD (E200K) mutation showed mild Proteinase K resistance when expressed in Drosophila. Adult Drosophila transgenic for FFI or fCJD variants of mouse or hamster PrP displayed a spontaneous decline in locomotor ability that increased in severity as the flies aged. Significantly, this mutant PrP-mediated neurotoxic fly phenotype was transferable to recipient Drosophila that expressed the wild-type form of the transgene. Collectively, our novel data are indicative of the spontaneous formation of a PrP-dependent neurotoxic phenotype in FFI- or CJD-PrP transgenic Drosophila and show that inherited human prion disease can be modelled in this invertebrate host.

Download Full-text

Differential Localization of RAC1 and RAC2 Reflects Their Specific Functions in Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v120.21.2302.2302 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2302-2302

Author(s):

Marta Ewa Capala ◽

Henny Maat ◽

Francesco Bonardi ◽

Edo Vellenga ◽

Jan Jacob Schuringa

Keyword(s):

Cell Cycle ◽

Plasma Membrane ◽

Molecular Mechanisms ◽

Specific Interaction ◽

Hematopoietic Cells ◽

Time Lapse ◽

Confocal Imaging ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Interaction Partners

Abstract Abstract 2302 Hematopoietic stem cells (HSCs) depend on the bone marrow niche to provide signals for their survival, quiescence and differentiation. Many of these microenvironmental signals converge on RAC GTPases. In the hematopoietic system, two members of the RAC family are expressed, RAC1 and RAC2. Although RAC1 and RAC2 share a very high sequence homology, specific functions of these proteins have been suggested. However, little has been revealed about the downstream effectors and molecular mechanisms. In this study, we used multiple approaches to gain insight into the molecular biology of RAC1 and RAC2 in normal and leukemic human HSCs. Firstly, GFP-tagged constructs of RAC1 and RAC2 were used to study localization of these proteins in CD34+/CD38−/Lin− HSCs. Time-lapse confocal imaging of living cells plated on stroma revealed that RAC1 was strongly enriched in the plasma membrane. In contrast, RAC2 localized predominantly in the cytoplasm of both resting and dividing HSCs, whereby localization changed dramatically when cells progressed from S to the G2 phase of the cell cycle. This very distinct localization pattern implied different functions of RAC1 and RAC2. Therefore, we specifically downregulated RAC1 and/or RAC2 to study the effects of their depletion in normal and BCR-ABL-transduced leukemic HSCs. In normal HSCs, simultaneous downregulation of RAC1 and RAC2 resulted in a modest but significant decrease in proliferation and progenitor frequencies in the long term stromal co-cultures. However, in BCR-ABL-transduced HSCs depletion of RAC2 alone, but not RAC1, was sufficient to induce a marked proliferative disadvantage, decreased progenitor frequency, reduced leukemic cobblestone formation and diminished replating capacity. To elucidate the mechanisms involved in the observed phenotypes, we employed an in vivo biotin labeling strategy of Avi-tagged RAC1 and RAC2 followed by pull down and mass-spectrometry to identify specific interaction partners of RAC1 and RAC2 in BCR-ABL-expressing hematopoietic cells. Several of the RAC1-specific interaction partners were annotated as plasma membrane proteins, involved in cell adhesion, cytoskeleton assembly and regulation of endocytosis. In contrast, RAC2-interacting proteins were cytoplasmic and involved in processes such as cell cycle progression, mitosis and regulation of apoptosis. Consistently with these findings, confocal time-lapse imaging of living hematopoietic cells revealed that pharmacological inhibition of RAC2 activity resulted in greatly decreased frequency of cell division. Moreover, the average division time was significantly extended upon RAC2 inhibition. Further functional characterization of RAC1 and RAC2-specific interactions is currently ongoing and will be discussed, but our data clearly indicate that distinct subcellular localization of RAC1 and RAC2 dictates their interaction with specific sets of proteins and consequently their specific functions in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Alternative splicing regulation of cell cycle genes by SPF45/SR140/CHERP complex controls cell proliferation

RNA ◽

10.1261/rna.078935.121 ◽

2021 ◽

pp. rna.078935.121

Author(s):

Elena Martin ◽

Claudia Vivori ◽

Malgorzata Rogalska ◽

Jorge Herrero ◽

Juan Valcarcel

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Alternative Splicing ◽

Cell Cycle Progression ◽

Molecular Mechanisms ◽

Splicing Factors ◽

Splicing Regulation ◽

Cancer Cell Proliferation ◽

Cycle Progression ◽

Regulation Of Cell Cycle

The regulation of pre-mRNA processing has important consequences for cell division and the control of cancer cell proliferation but the underlying molecular mechanisms remain poorly understood. We report that three splicing factors, SPF45, SR140 and CHERP form a tight physical and functionally coherent complex that regulates a variety of alternative splicing events, frequently by repressing short exons flanked by suboptimal 3' splice sites. These comprise alternative exons embedded in genes with important functions in cell cycle progression, including the G2/M key regulator FOXM1 and the spindle regulator SPDL1. Knockdown of either of the three factors leads to G2/M arrest and to enhanced apoptosis in HeLa cells. Promoting the changes in FOXM1 or SPDL1 splicing induced by SPF45/SR140/CHERP knockdown partially recapitulate the effects on cell growth, arguing that the complex orchestrates a program of alternative splicing necessary for efficient cell proliferation.

Download Full-text

Ion channnels and transporters in cancer. 5. Ion channels in control of cancer and cell apoptosis

AJP Cell Physiology ◽

10.1152/ajpcell.00249.2011 ◽

2011 ◽

Vol 301 (6) ◽

pp. C1281-C1289 ◽

Cited By ~ 54

Author(s):

V'yacheslav Lehen'kyi ◽

George Shapovalov ◽

Roman Skryma ◽

Natalia Prevarskaya

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Plasma Membrane ◽

Ion Channels ◽

Programmed Cell Death ◽

Tissue Homeostasis ◽

Death Rates ◽

Cellular Processes ◽

Regulation Of Cell Cycle

Ion channels contribute to virtually all basic cellular processes, including such crucial ones for maintaining tissue homeostasis as proliferation, differentiation, and apoptosis. The involvement of ion channels in regulation of programmed cell death, or apoptosis, has been known for at least three decades based on observation that classical blockers of ion channels can influence cell death rates, prolonging or shortening cell survival. Identification of the central role of these channels in regulation of cell cycle and apoptosis as well as the recent discovery that the expression of ion channels is not limited solely to the plasma membrane, but may also include membranes of internal compartments, has led researchers to appreciate the pivotal role of ion channels plays in development of cancer. This review focuses on the aspects of programmed cell death influenced by various ion channels and how dysfunctions and misregulations of these channels may affect the development and progression of different cancers.

Download Full-text

Differential Localization Of RAC1 and RAC2 Reflects Their Specific Functions In Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v122.21.2892.2892 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2892-2892

Author(s):

Marta Ewa Capala ◽

Francesco Bonardi ◽

Henny Maat ◽

Edo Vellenga ◽

Jan Jacob Schuringa

Keyword(s):

Cell Cycle ◽

Plasma Membrane ◽

Molecular Mechanisms ◽

Specific Interaction ◽

Hematopoietic Cells ◽

Confocal Imaging ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Interaction Partners

Abstract Hematopoietic stem cells (HSCs) depend on the bone marrow niche to provide signals for their survival, quiescence and differentiation. Many of these microenvironmental signals converge on RAC GTPases. In the hematopoietic system, two members of the RAC family are expressed, RAC1 and RAC2. Although RAC1 and RAC2 share a very high sequence homology, specific functions of these proteins have been suggested. However, little has been revealed about the downstream effectors and molecular mechanisms. In this study, we used multiple approaches to gain insight into the molecular biology of RAC1 and RAC2 in normal and leukemic human HSCs. Firstly, GFP-tagged constructs of RAC1 and RAC2 were used to study localization of these proteins in CD34+/CD38-/Lin- HSCs. Time-lapse confocal imaging of living cells plated on stroma revealed that RAC1 was strongly enriched in the plasma membrane. In contrast, RAC2 localized predominantly in the cytoplasm of both resting and dividing HSCs, whereby localization changed dramatically when cells progressed from S to the G2 phase of the cell cycle. This very distinct localization pattern implied different functions of RAC1 and RAC2. Therefore, we specifically downregulated RAC1 and/or RAC2 to study the effects of their depletion in normal and BCR-ABL-transduced leukemic HSCs. In normal HSCs, simultaneous downregulation of RAC1 and RAC2 resulted in a modest but significant decrease in proliferation and progenitor frequencies in the long term stromal co-cultures. However, in BCR-ABL-transduced HSCs depletion of RAC2 alone, but not RAC1, was sufficient to induce a marked proliferative disadvantage, decreased progenitor frequency, reduced leukemic cobblestone formation and diminished replating capacity, indicative for reduced self-renewal. Consistently, the frequency of long-term culture initiating leukemic cells was markedly reduced upon RAC2 downregulation. To elucidate the mechanisms involved in the observed phenotypes, we employed an in vivo biotin labeling strategy of Avi-tagged RAC1 and RAC2 followed by pull down and mass-spectrometry to identify specific interaction partners of RAC1 and RAC2 in BCR-ABL-expressing hematopoietic cells. Several of the RAC1-specific interaction partners were annotated as plasma membrane proteins, involved in cell adhesion, cytoskeleton assembly and regulation of endocytosis. In contrast, RAC2-interacting proteins were cytoplasmic or mitochondria-associated, and involved in processes such as cell cycle progression and regulation of apoptosis. Consistently, the proportion of dividing cells was decreased in RAC2-depleted BCR-ABL leukemic cobblestones coinciding with an increased apoptosis. Finally, a marked decrease in mitochondrial membrane potential was observed upon RAC2 but not RAC1 downregulation pointing to mitochondrial dysfunction as the initiating event of the apoptotic response. Moreover, preliminary electron microscopy data suggest that this functional change may be paralleled by structural aberrations of mitochondria. Further functional characterization of RAC1 and RAC2-specific interactions is currently ongoing and will be discussed, but our data clearly indicate that distinct subcellular localization of RAC1 and RAC2 dictates their interaction with specific sets of proteins and consequently their specific functions in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Cellular and Molecular Mechanisms of Prion Disease

Annual Review of Pathology Mechanisms of Disease ◽

10.1146/annurev-pathmechdis-012418-013109 ◽

2019 ◽

Vol 14 (1) ◽

pp. 497-516 ◽

Cited By ~ 15

Author(s):

Christina J. Sigurdson ◽

Jason C. Bartz ◽

Markus Glatzel

Keyword(s):

Prion Protein ◽

Prion Disease ◽

Molecular Mechanisms ◽

Prion Diseases ◽

Neuronal Loss ◽

Cellular Prion Protein ◽

Future Research ◽

Spongiform Encephalopathy ◽

Disease Epidemics ◽

Infectious Proteins

Prion diseases are rapidly progressive, incurable neurodegenerative disorders caused by misfolded, aggregated proteins known as prions, which are uniquely infectious. Remarkably, these infectious proteins have been responsible for widespread disease epidemics, including kuru in humans, bovine spongiform encephalopathy in cattle, and chronic wasting disease in cervids, the latter of which has spread across North America and recently appeared in Norway and Finland. The hallmark histopathological features include widespread spongiform encephalopathy, neuronal loss, gliosis, and deposits of variably sized aggregated prion protein, ranging from small, soluble oligomers to long, thin, unbranched fibrils, depending on the disease. Here, we explore recent advances in prion disease research, from the function of the cellular prion protein to the dysfunction triggering neurotoxicity, as well as mechanisms underlying prion spread between cells. We also highlight key findings that have revealed new therapeutic targets and consider unanswered questions for future research.

Download Full-text

PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo

The Journal of Cell Biology ◽

10.1083/jcb.201312029 ◽

2014 ◽

Vol 205 (4) ◽

pp. 447-455 ◽

Cited By ~ 18

Author(s):

Laura Benkemoun ◽

Catherine Descoteaux ◽

Nicolas T. Chartier ◽

Lionel Pintard ◽

Jean-Claude Labbé

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Cell Cycle Progression ◽

Molecular Mechanisms ◽

Control Cell ◽

Cycle Duration ◽

Cell Stage ◽

C Elegans ◽

Stage Embryo ◽

Regulation Of Cell Cycle

Regulation of cell cycle duration is critical during development, yet the underlying molecular mechanisms are still poorly understood. The two-cell stage Caenorhabditis elegans embryo divides asynchronously and thus provides a powerful context in which to study regulation of cell cycle timing during development. Using genetic analysis and high-resolution imaging, we found that deoxyribonucleic acid (DNA) replication is asymmetrically regulated in the two-cell stage embryo and that the PAR-4 and PAR-1 polarity proteins dampen DNA replication dynamics specifically in the posterior blastomere, independently of regulators previously implicated in the control of cell cycle timing. Our results demonstrate that accurate control of DNA replication is crucial during C. elegans early embryonic development and further provide a novel mechanism by which PAR proteins control cell cycle progression during asynchronous cell division.

Download Full-text

Cell cycle– and swelling-induced activation of a Caenorhabditis elegans ClC channel is mediated by CeGLC-7α/β phosphatases

The Journal of Cell Biology ◽

10.1083/jcb.200204142 ◽

2002 ◽

Vol 158 (3) ◽

pp. 435-444 ◽

Cited By ~ 37

Author(s):

Eric Rutledge ◽

Jerod Denton ◽

Kevin Strange

Keyword(s):

Cell Cycle ◽

Caenorhabditis Elegans ◽

Molecular Mechanisms ◽

Cell Swelling ◽

Phosphatase Inhibitors ◽

C Elegans ◽

Physiological Processes ◽

Cycle Dependent ◽

Regulation Of Cell Cycle

ClC voltage-gated anion channels have been identified in bacteria, yeast, plants, and animals. The biophysical and structural properties of ClCs have been studied extensively, but relatively little is known about their precise physiological functions. Furthermore, virtually nothing is known about the signaling pathways and molecular mechanisms that regulate channel activity. The nematode Caenorhabditis elegans provides significant experimental advantages for characterizing ion channel function and regulation. We have shown previously that the ClC Cl− channel homologue CLH-3 is expressed in C. elegans oocytes, and that it is activated during meiotic maturation and by cell swelling. We demonstrate here that depletion of intracellular ATP or removal of Mg2+, experimental maneuvers that inhibit kinase function, constitutively activate CLH-3. Maturation- and swelling-induced channel activation are inhibited by type 1 serine/threonine phosphatase inhibitors. RNA interference studies demonstrated that the type 1 protein phosphatases CeGLC-7α and β, both of which play essential regulatory roles in mitotic and meiotic cell cycle events, mediate CLH-3 activation. We have suggested previously that CLH-3 and mammalian ClC-2 are orthologues that play important roles in heterologous cell–cell interactions, intercellular communication, and regulation of cell cycle–dependent physiological processes. Consistent with this hypothesis, we show that heterologously expressed rat ClC-2 is also activated by serine/threonine dephosphorylation, suggesting that the two channels have common regulatory mechanisms.

Download Full-text

Differential Accumulation of Misfolded Prion Strains in Natural Hosts of Prion Diseases

Viruses ◽

10.3390/v13122453 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2453

Author(s):

Zoe J. Lambert ◽

Justin J. Greenlee ◽

Eric D. Cassmann ◽

M. Heather West Greenlee

Keyword(s):

Prion Protein ◽

Prion Disease ◽

Prion Diseases ◽

Brain Regions ◽

Cellular Prion Protein ◽

Transmissible Spongiform Encephalopathies ◽

Spongiform Encephalopathy ◽

Prion Strains ◽

Natural Hosts ◽

Different Strains

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of neurodegenerative protein misfolding diseases that invariably cause death. TSEs occur when the endogenous cellular prion protein (PrPC) misfolds to form the pathological prion protein (PrPSc), which templates further conversion of PrPC to PrPSc, accumulates, and initiates a cascade of pathologic processes in cells and tissues. Different strains of prion disease within a species are thought to arise from the differential misfolding of the prion protein and have different clinical phenotypes. Different strains of prion disease may also result in differential accumulation of PrPSc in brain regions and tissues of natural hosts. Here, we review differential accumulation that occurs in the retinal ganglion cells, cerebellar cortex and white matter, and plexuses of the enteric nervous system in cattle with bovine spongiform encephalopathy, sheep and goats with scrapie, cervids with chronic wasting disease, and humans with prion diseases. By characterizing TSEs in their natural host, we can better understand the pathogenesis of different prion strains. This information is valuable in the pursuit of evaluating and discovering potential biomarkers and therapeutics for prion diseases.

Download Full-text