scholarly journals Altered ER–mitochondria contact impacts mitochondria calcium homeostasis and contributes to neurodegeneration in vivo in disease models

2018 ◽  
Vol 115 (38) ◽  
pp. E8844-E8853 ◽  
Author(s):  
Kyu-Sun Lee ◽  
Sungun Huh ◽  
Seongsoo Lee ◽  
Zhihao Wu ◽  
Ae-Kyeong Kim ◽  
...  
Keyword(s):  
Neurological Diseases ◽  
Cell Types ◽  
Mitochondrial Morphology ◽  
Loss Of Function ◽  
Neuronal Function ◽  
Independent Manner ◽  
Lrrk2 G2019s ◽  
Mutant Phenotypes ◽  
Cellular Compartments

Calcium (Ca2+) homeostasis is essential for neuronal function and survival. Altered Ca2+ homeostasis has been consistently observed in neurological diseases. How Ca2+ homeostasis is achieved in various cellular compartments of disease-relevant cell types is not well understood. Here we show in Drosophila Parkinson’s disease (PD) models that Ca2+ transport from the endoplasmic reticulum (ER) to mitochondria through the ER–mitochondria contact site (ERMCS) critically regulates mitochondrial Ca2+ (mito-Ca2+) homeostasis in dopaminergic (DA) neurons, and that the PD-associated PINK1 protein modulates this process. In PINK1 mutant DA neurons, the ERMCS is strengthened and mito-Ca2+ level is elevated, resulting in mitochondrial enlargement and neuronal death. Miro, a well-characterized component of the mitochondrial trafficking machinery, mediates the effects of PINK1 on mito-Ca2+ and mitochondrial morphology, apparently in a transport-independent manner. Miro overexpression mimics PINK1 loss-of-function effect, whereas inhibition of Miro or components of the ERMCS, or pharmacological modulation of ERMCS function, rescued PINK1 mutant phenotypes. Mito-Ca2+ homeostasis is also altered in the LRRK2-G2019S model of PD and the PAR-1/MARK model of neurodegeneration, and genetic or pharmacological restoration of mito-Ca2+ level is beneficial in these models. Our results highlight the importance of mito-Ca2+ homeostasis maintained by Miro and the ERMCS to mitochondrial physiology and neuronal integrity. Targeting this mito-Ca2+ homeostasis pathway holds promise for a therapeutic strategy for neurodegenerative diseases.

scholarly journals A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Sonal Nagarkar-Jaiswal ◽  
Pei-Tseng Lee ◽  
Megan E Campbell ◽  
Kuchuan Chen ◽  
Stephanie Anguiano-Zarate ◽  
...  
Keyword(s):  
Null Mutant ◽  
Loss Of Function ◽  
Severe Loss ◽  
New Strategy ◽  
Mutant Phenotypes

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

scholarly journals Assessment of phagocytic activity in live macrophages-tumor cells co-cultures by Confocal and Nomarski Microscopy

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Dalia Martinez-Marin ◽  
Courtney Jarvis ◽  
Thomas Nelius ◽  
Stéphanie Filleur
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Phagocytic Activity ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Functional Assay ◽  
Loss Of Function ◽  
Multiple Cancer

Abstract Macrophages have been recognized as the main inflammatory component of the tumor microenvironment. Although often considered as beneficial for tumor growth and disease progression, tumor-associated macrophages have also been shown to be detrimental to the tumor depending on the tumor microenvironment. Therefore, understanding the molecular interactions between macrophages and tumor cells in relation to macrophages functional activities such as phagocytosis is critical for a better comprehension of their tumor-modulating action. Still, the characterization of these molecular mechanisms in vivo remains complicated due to the extraordinary complexity of the tumor microenvironment and the broad range of tumor-associated macrophage functions. Thus, there is an increasing demand for in vitro methodologies to study the role of cell–cell interactions in the tumor microenvironment. In the present study, we have developed live co-cultures of macrophages and human prostate tumor cells to assess the phagocytic activity of macrophages using a combination of Confocal and Nomarski Microscopy. Using this model, we have emphasized that this is a sensitive, measurable, and highly reproducible functional assay. We have also highlighted that this assay can be applied to multiple cancer cell types and used as a selection tool for a variety of different types of phagocytosis agonists. Finally, combining with other studies such as gain/loss of function or signaling studies remains possible. A better understanding of the interactions between tumor cells and macrophages may lead to the identification of new therapeutic targets against cancer.

scholarly journals Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy

2015 ◽  
Vol 113 (1) ◽  
pp. 182-187 ◽  
Author(s):  
Christina H. Eng ◽  
Zuncai Wang ◽  
Diane Tkach ◽  
Lourdes Toral-Barza ◽  
Savuth Ugwonali ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Kinase Inhibitors ◽  
Cell Types ◽  
Loss Of Function ◽  
Growth And Survival ◽  
Oncogenic Mutations ◽  
Cellular Context ◽  
Genetic Stratification

Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic loss-of-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagy-related 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy.

scholarly journals Essential Roles in Development and Pigmentation for the Drosophila Copper Transporter DmATP7

2006 ◽  
Vol 17 (1) ◽  
pp. 475-484 ◽  
Author(s):  
Melanie Norgate ◽  
Esther Lee ◽  
Adam Southon ◽  
Ashley Farlow ◽  
Philip Batterham ◽  
...  
Keyword(s):  
Copper Homeostasis ◽  
Cellular Level ◽  
Loss Of Function ◽  
Neuronal Function ◽  
Copper Transporter ◽  
Disease Phenotypes ◽  
In Vivo Analysis ◽  
Cellular Copper ◽  
P Type

Defects in the mammalian Menkes and Wilson copper transporting P-type ATPases cause severe copper homeostasis disease phenotypes in humans. Here, we find that DmATP7, the sole Drosophila orthologue of the Menkes and Wilson genes, is vital for uptake of copper in vivo. Analysis of a DmATP7 loss-of-function allele shows that DmATP7 is essential in embryogenesis, early larval development, and adult pigmentation and is probably required for copper uptake from the diet. These phenotypes are analogous to those caused by mutation in the mouse and human Menkes genes, suggesting that like Menkes, DmATP7 plays at least two roles at the cellular level: delivering copper to cuproenzymes required for pigmentation and neuronal function and removing excess cellular copper via facilitated efflux. DmATP7 displays a dynamic and unexpected expression pattern in the developing embryo, implying novel functions for this copper pump and the lethality observed in DmATP7 mutant flies is the earliest seen for any copper homeostasis gene.

scholarly journals Perspectives for Applying G-Quadruplex Structures in Neurobiology and Neuropharmacology

2019 ◽  
Vol 20 (12) ◽  
pp. 2884 ◽  
Author(s):  
Sefan Asamitsu ◽  
Masayuki Takeuchi ◽  
Susumu Ikenoshita ◽  
Yoshiki Imai ◽  
Hirohito Kashiwagi ◽  
...  
Keyword(s):  
Neurological Disorders ◽  
Neurological Diseases ◽  
Secondary Structures ◽  
Neuronal Function ◽  
Double Stranded Rna ◽  
Quadruplex Dna ◽  
Functional Roles ◽  
G Quadruplex ◽  
Dna Secondary Structures

The most common form of DNA is a right-handed helix or the B-form DNA. DNA can also adopt a variety of alternative conformations, non-B-form DNA secondary structures, including the DNA G-quadruplex (DNA-G4). Furthermore, besides stem-loops that yield A-form double-stranded RNA, non-canonical RNA G-quadruplex (RNA-G4) secondary structures are also observed. Recent bioinformatics analysis of the whole-genome and transcriptome obtained using G-quadruplex–specific antibodies and ligands, revealed genomic positions of G-quadruplexes. In addition, accumulating evidence pointed to the existence of these structures under physiologically- and pathologically-relevant conditions, with functional roles in vivo. In this review, we focused on DNA-G4 and RNA-G4, which may have important roles in neuronal function, and reveal mechanisms underlying neurological disorders related to synaptic dysfunction. In addition, we mention the potential of G-quadruplexes as therapeutic targets for neurological diseases.

Role of Notch Signaling in Hematopoietic Stem Cell Function

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-15-SCI-15
Author(s):  
Lluis Espinosa ◽  
Anna Bigas
Keyword(s):  
Stem Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Notch Pathway ◽  
Cell Types ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
On Chip ◽  
Stem Cell Populations

Abstract Abstract SCI-15 The Notch pathway controls the generation of different cell types in most tissues including blood, and dysregulation of this pathway is strongly associated with oncogenic processes. In many systems, Notch is also required for the maintenance of the stem cell populations. However, in the adult hematopoietic system this link between Notch and stemness has not been established. Instead, work of several groups, including ours, has clearly demonstrated that Notch has a prominent role in the generation of hematopoietic stem cells (HSC) during embryonic development. Although the first wave of blood cells appears in the mouse embryo around day 7.5 of development and is independent of Notch function, embryonic HSC are formed around day 10 of development from endothelial-like progenitors that reside in the embryonic aorta surrounded by the gonad and mesonephros, also called AGM region. By analyzing different Notch pathway mutant mouse embryos, we have demonstrated the involvement of the Jagged1-Notch1-GATA2 axis in this event. However, the formal demonstration that Notch regulates the GATA2 gene during HSC generation is still lacking. We have now found that GATA2 is a direct Notch target in vivo during embryonic HSC generation. However, whereas Notch positively activates GATA2 transcription in the HSC precursors, it simultaneously activates hes1 transcription, which acts a repressor of the same GATA2 gene. This finding directly implicates hes1 in the regulation of HSC development although further studies using loss-of-function mutant embryos are still needed. Altogether, our results indicate that both Notch and hes1 are required to finely regulate the levels, distribution, and likely the timing of GATA2 expression through an incoherent feed-forward loop. In parallel, we have identified other downstream targets of Notch in the AGM region by ChIP-on-chip and expression microarray analysis that we are currently characterizing. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Neural plate targeting by in utero nanoinjection (NEPTUNE) reveals a role for Sptbn2 in neurulation and abdominal wall closure

2020 ◽  
Author(s):  
Katrin Mangold ◽  
Jan Mašek ◽  
Jingyan He ◽  
Urban Lendahl ◽  
Elaine Fuchs ◽  
...  
Keyword(s):  
Cost Effective ◽  
Cell Types ◽  
Neural Plate ◽  
Spinocerebellar Ataxia Type ◽  
Loss Of Function ◽  
Conditional Expression ◽  
Cost Effective Technique ◽  
The Brain

ABSTRACTGene variants associated with disease are efficiently identified with whole genome sequencing or GWAS, but validation in vivo lags behind. We developed NEPTUNE (neural plate targeting by in utero nanoinjection), to rapidly and flexibly introduce gene expression-modifying viruses to the embryonic murine neural plate prior to neurulation, to target the future adult nervous system. Stable integration in >95% of cells in the brain enabled long-term gain- or loss-of-function, and conditional expression was achieved using mini-promotors for cell types of interest. Using NEPTUNE, we silenced Sptbn2, a gene associated with Spinocerebellar ataxia type 5 (SCA5) in humans. Silencing of Sptbn2 induced severe neural tube defects and embryo resorption, suggesting that SPTBN2 in-frame and missense deletions in SCA5 reflect hypomorphic or neomorphic functions, not loss of function. In conclusion, NEPTUNE offers a novel, rapid and cost-effective technique to test gene function in brain development, and can reveal loss of function phenotypes incompatible with life.

scholarly journals FMRP binding to a ranked subset of long genes is revealed by coupled CLIP and TRAP in specific neuronal cell types

2019 ◽  
Author(s):  
Sarah J. Van Driesche ◽  
Kirsty Sawicka ◽  
Chaolin Zhang ◽  
Sharon K.Y. Hung ◽  
Christopher Y. Park ◽  
...  
Keyword(s):  
Purkinje Cells ◽  
Fragile X ◽  
Neuronal Cell ◽  
Transcript Abundance ◽  
Cell Types ◽  
Loss Of Function ◽  
Ip3 Receptor ◽  
Mrna Targets ◽  
Ranked List

SummaryLoss of function of the Fragile X Mental Retardation Protein (FMRP) in human Fragile X Syndrome (FXS) and in model organisms results in phenotypes of abnormal neuronal structure and dynamics, synaptic function and connectivity which may contribute to a state of neuronal, circuit and organism hyperexcitability. Previousin vivoidentification of FMRP association with specific mRNA targets in mouse brain revealed that FMRP regulates the translation of a large fraction of the synaptic proteome in both pre- and post-synaptic compartments as well as many transcription factors and chromatin modifying proteins. However, it was not previously possible to determine the ratio of FMRP binding to transcript abundance due to the complexity of different neuronal cell types in whole brain. Moreover, it has been difficult to link the translational regulation of specific targets to model phenotypes or human symptoms. For example, loss-of-function of FMRP in the Purkinje cells of the cerebellum results in three cell autonomous phenotypes related to learning and memory, including enhanced mGluR-LTD at parallel fiber synapses, altered dendritic spines and behavioral deficits in a eyeblink-conditioning learning paradigm shared by human FXS patients. The molecular basis for these and related human Fragile X phenotypes is unknown. To address these critical issues we have developed a new mouse model (theFmr1cTAG mouse) in which endogenous FMRP can be conditionally tagged for RNA:protein crosslinking and immunoprecipitation (CLIP) identification of the RNAs with which it interactsin vivo. We used theFmr1cTAG mouse to quantitatively evaluate FMRP-mRNA association in Purkinje and cerebellar granule neurons which together comprise the parallel-fiber synapse. We calculated a stoichiometrically ranked list of FMRP RNA binding events by normalizing to ribosome-associated transcript abundance determined by TRAP-seq, and now definitively find that FMRP associates with specific sets of mRNAs which differ between the two cell types. In Purkinje cells, many components of the mGluR signaling pathway are FMRP targets including the top-ranked Purkinje cell mRNAItpr1, encoding the IP3 receptor, the function of which is critical to proper mGluR-dependent synaptic plasticity. In sum, this novel approach provides the first ranked list of FMRP target mRNAs and further reveals that FMRP regulates a specific set of long neural genes related to relevant cell autonomous phenotypes.HighlightsWe have created a mouse model in which endogenous FMRP can be conditionally tagged.Using tag-specific CLIP we describe ranked and specific sets ofin vivoFMRP mRNA targets in two types of neurons.This ranking was used to reveal that FMRP regulates mRNAs with long coding sequences.FMRP mRNA targets in Purkinje cells, including the top-ranked IP3 receptor, are related to cell-autonomous Fragile X phenotypes.We have updated our previous list of whole mouse brain FMRP mRNA targets with more replicates, deeper sequencing and improved analysisThe use of tagged FMRP in less abundant cell populations allowed identification of novel mRNA targets missed in a whole brain analysis

scholarly journals Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

2021 ◽  
Author(s):  
Sruti Rayaprolu ◽  
Sara Bitarafan ◽  
Ranjita Betarbet ◽  
Sydney N Sunna ◽  
Lihong Cheng ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Neurological Diseases ◽  
Neuronal Cell ◽  
Cell Types ◽  
Proteomic Profiling ◽  
Cell Type ◽  
Specific Expression ◽  
Cell Type Specific ◽  
The Brain

Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain.

scholarly journals Evolutionary conservation and divergence of human brain co-expression networks

2020 ◽  
Author(s):  
WG Pembroke ◽  
CL Hartl ◽  
DH Geschwind
Keyword(s):  
Human Brain ◽  
Mouse Models ◽  
Disease Modeling ◽  
Disease Risk ◽  
Sequence Divergence ◽  
Cell Types ◽  
Regulatory Sequence ◽  
Loss Of Function ◽  
Regional Patterns

AbstractMouse models have allowed for the direct interrogation of genetic effects on molecular, physiological and behavioral brain phenotypes. However, it is unknown to what extent neurological or psychiatric traits may be human or primate-specific and therefore, which components can be faithfully recapitulated in mouse models. We identify robust co-expression modules reflecting whole brain and regional patterns of gene expression and compare conservation of co-expression in 116 independent data sets derived from human, mouse and non-human primate representing more than 15,000 total samples. We observe greater co-expression changes occurring on the human lineage than mouse, and substantial regional variation that highlights cerebral cortex as the most diverged region. Cell type specific modules are the most divergent across the brain, compared with those that represent basic metabolic processes. Among these, glia are the most divergent, three times that of neurons. We show that regulatory sequence divergence explains a significant fraction of co-expression divergence. Similarly, protein coding sequence constraint parallels co-expression conservation, such that genes with loss of function intolerance are enriched in neuronal, rather than glial modules. We also identify dozens of human disease risk genes, such as COMT, PSEN-1, LRRK2, and SNCA, with highly divergent co-expression between mouse and primates or human. We show that 3D human brain organoids recapitulate in vivo co-expression modules representing several human cell types, which along with our analysis of human-mouse disease gene divergence, serve as a foundational resource to guide disease modeling and its interpretation.

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