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1.
Mol Neurodegener ; 19(1): 50, 2024 Jun 20.
Article in English | MEDLINE | ID: mdl-38902734

ABSTRACT

BACKGROUND: The key pathological signature of ALS/ FTLD is the mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm. However, TDP-43 gain of function in the cytoplasm is still poorly understood since TDP-43 animal models recapitulating mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm are missing. METHODS: CRISPR/Cas9 technology was used to generate a zebrafish line (called CytoTDP), that mis-locates endogenous TDP-43 from the nucleus to the cytoplasm. Phenotypic characterization of motor neurons and the neuromuscular junction was performed by immunostaining, microglia were immunohistochemically localized by whole-mount tissue clearing and muscle ultrastructure was analyzed by scanning electron microscopy. Behavior was investigated by video tracking and quantitative analysis of swimming parameters. RNA sequencing was used to identify mis-regulated pathways with validation by molecular analysis. RESULTS: CytoTDP fish have early larval phenotypes resembling clinical features of ALS such as progressive motor defects, neurodegeneration and muscle atrophy. Taking advantage of zebrafish's embryonic development that solely relys on yolk usage until 5 days post fertilization, we demonstrated that microglia proliferation and activation in the hypothalamus is independent from food intake. By comparing CytoTDP to a previously generated TDP-43 knockout line, transcriptomic analyses revealed that mis-localization of endogenous TDP-43, rather than TDP-43 nuclear loss of function, leads to early onset metabolic dysfunction. CONCLUSIONS: The new TDP-43 model mimics the ALS/FTLD hallmark of progressive motor dysfunction. Our results suggest that functional deficits of the hypothalamus, the metabolic regulatory center, might be the primary cause of weight loss in ALS patients. Cytoplasmic gain of function of endogenous TDP-43 leads to metabolic dysfunction in vivo that are reminiscent of early ALS clinical non-motor metabolic alterations. Thus, the CytoTDP zebrafish model offers a unique opportunity to identify mis-regulated targets for therapeutic intervention early in disease progression.


Subject(s)
Amyotrophic Lateral Sclerosis , DNA-Binding Proteins , Disease Models, Animal , Motor Neurons , Zebrafish Proteins , Zebrafish , Animals , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Motor Neurons/metabolism , Motor Neurons/pathology , Zebrafish Proteins/metabolism , Zebrafish Proteins/genetics , Animals, Genetically Modified , Neuromuscular Junction/metabolism , Neuromuscular Junction/pathology
2.
Front Cell Dev Biol ; 11: 1169962, 2023.
Article in English | MEDLINE | ID: mdl-37384248

ABSTRACT

Aggregation of the Tar DNA-binding protein of 43 kDa (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia and likely contributes to disease by loss of nuclear function. Analysis of TDP-43 function in knockout zebrafish identified an endothelial directional migration and hypersprouting phenotype during development prior lethality. In human umbilical vein cells (HUVEC) the loss of TDP-43 leads to hyperbranching. We identified elevated expression of FIBRONECTIN 1 (FN1), the VASCULAR CELL ADHESION MOLECULE 1 (VCAM1), as well as their receptor INTEGRIN α4ß1 (ITGA4B1) in HUVEC cells. Importantly, reducing the levels of ITGA4, FN1, and VCAM1 homologues in the TDP-43 loss-of-function zebrafish rescues the angiogenic defects indicating the conservation of human and zebrafish TDP-43 function during angiogenesis. Our study identifies a novel pathway regulated by TDP-43 important for angiogenesis during development.

3.
Mol Metab ; 74: 101748, 2023 08.
Article in English | MEDLINE | ID: mdl-37290673

ABSTRACT

OBJECTIVE: Cancer cells convert more glucose into lactate than healthy cells, what contributes to their growth advantage. Pyruvate kinase (PK) is a key rate limiting enzyme in this process, what makes it a promising potential therapeutic target. However, currently it is still unclear what consequences the inhibition of PK has on cellular processes. Here, we systematically investigate the consequences of PK depletion for gene expression, histone modifications and metabolism. METHODS: Epigenetic, transcriptional and metabolic targets were analysed in different cellular and animal models with stable knockdown or knockout of PK. RESULTS: Depleting PK activity reduces the glycolytic flux and causes accumulation of glucose-6-phosphate (G6P). Such metabolic perturbation results in stimulation of the activity of a heterodimeric pair of transcription factors MondoA and MLX but not in a major reprogramming of the global H3K9ac and H3K4me3 histone modification landscape. The MondoA:MLX heterodimer upregulates expression of thioredoxin-interacting protein (TXNIP) - a tumour suppressor with multifaceted anticancer activity. This effect of TXNIP upregulation extends beyond immortalised cancer cell lines and is applicable to multiple cellular and animal models. CONCLUSIONS: Our work shows that actions of often pro-tumorigenic PK and anti-tumorigenic TXNIP are tightly linked via a glycolytic intermediate. We suggest that PK depletion stimulates the activity of MondoA:MLX transcription factor heterodimers and subsequently, increases cellular TXNIP levels. TXNIP-mediated inhibition of thioredoxin (TXN) can reduce the ability of cells to scavenge reactive oxygen species (ROS) leading to the oxidative damage of cellular structures including DNA. These findings highlight an important regulatory axis affecting tumour suppression mechanisms and provide an attractive opportunity for combination cancer therapies targeting glycolytic activity and ROS-generating pathways.


Subject(s)
Neoplasms , Pyruvate Kinase , Animals , Pyruvate Kinase/genetics , Reactive Oxygen Species , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Thioredoxins/chemistry , Thioredoxins/metabolism
4.
Mol Neurodegener ; 12(1): 6, 2017 01 14.
Article in English | MEDLINE | ID: mdl-28088213

ABSTRACT

BACKGROUND: The most frequent genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) is the expansion of a GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 (C9orf72) locus. The pathological hallmarks observed in C9orf72 repeat expansion carriers are the formation of RNA foci and deposition of dipeptide repeat (DPR) proteins derived from repeat associated non-ATG (RAN) translation. Currently, it is unclear whether formation of RNA foci, DPR translation products, or partial loss of C9orf72 predominantly drive neurotoxicity in vivo. By using a transgenic approach in zebrafish we address if the most frequently found DPR in human ALS/FTLD brain, the poly-Gly-Ala (poly-GA) protein, is toxic in vivo. METHOD: We generated several transgenic UAS responder lines that express either 80 repeats of GGGGCC alone, or together with a translation initiation ATG codon forcing the translation of GA80-GFP protein upon crossing to a Gal4 driver. The GGGGCC repeat and GA80 were fused to green fluorescent protein (GFP) lacking a start codon to monitor protein translation by GFP fluorescence. RESULTS: Zebrafish transgenic for the GGGGCC repeat lacking an ATG codon showed very mild toxicity in the absence of poly-GA. However, strong toxicity was induced upon ATG initiated expression of poly-GA, which was rescued by injection of an antisense morpholino interfering with start codon dependent poly-GA translation. This morpholino only interferes with GA80-GFP translation without affecting repeat transcription, indicating that the toxicity is derived from GA80-GFP. CONCLUSION: These novel transgenic C9orf72 associated repeat zebrafish models demonstrate poly-GA toxicity in zebrafish. Reduction of poly-GA protein rescues toxicity validating this therapeutic approach to treat C9orf72 repeat expansion carriers. These novel animal models provide a valuable tool for drug discovery to reduce DPR associated toxicity in ALS/FTLD patients with C9orf72 repeat expansions.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Frontotemporal Lobar Degeneration/genetics , Open Reading Frames , Peptides/toxicity , Amyotrophic Lateral Sclerosis/pathology , Animals , Animals, Genetically Modified , Blotting, Western , Dinucleotide Repeats , Disease Models, Animal , Frontotemporal Lobar Degeneration/pathology , Immunohistochemistry , In Situ Hybridization, Fluorescence , Polymerase Chain Reaction , Polymers , Zebrafish
5.
Methods Mol Biol ; 1254: 341-50, 2015.
Article in English | MEDLINE | ID: mdl-25431076

ABSTRACT

The CRISPR /Cas system identified in archaea has been adopted and optimized for genome editing purposes in zebrafish. In vitro transcribed guide RNA and Cas9 mRNA are microinjected into fertilized zebrafish embryos to edit the zebrafish genome. Here, we describe how to design a gRNA, a fast method for in vitro transcription of gRNA from oligonucleotides , microinjection into fertilized zebrafish embryos, and a PCR -based restriction fragment length assay to identify mutations at the gRNA target site.


Subject(s)
CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems , Gene Editing/methods , RNA, Guide, Kinetoplastida/genetics , Zebrafish/genetics , Animals , Genome , Models, Animal , Mutation
6.
Development ; 140(24): 4982-7, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24257628

ABSTRACT

Gene modifications in animal models have been greatly facilitated through the application of targeted genome editing tools. The prokaryotic CRISPR/Cas9 type II genome editing system has recently been applied in cell lines and vertebrates. However, we still have very limited information about the efficiency of mutagenesis, germline transmission rates and off-target effects in genomes of model organisms. We now demonstrate that CRISPR/Cas9 mutagenesis in zebrafish is highly efficient, reaching up to 86.0%, and is heritable. The efficiency of the CRISPR/Cas9 system further facilitated the targeted knock-in of a protein tag provided by a donor oligonucleotide with knock-in efficiencies of 3.5-15.6%. Mutation rates at potential off-target sites are only 1.1-2.5%, demonstrating the specificity of the CRISPR/Cas9 system. The ease and efficiency of the CRISPR/Cas9 system with limited off-target effects make it a powerful genome engineering tool for in vivo studies.


Subject(s)
Bacterial Proteins/genetics , CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems , Endonucleases/genetics , Gene Editing/methods , Zebrafish/genetics , Animals , Base Sequence , CRISPR-Associated Protein 9 , Gene Knock-In Techniques , Genome , Mutagenesis , Oligonucleotides, Antisense/genetics , Sequence Analysis, DNA
7.
Proc Natl Acad Sci U S A ; 110(13): 4986-91, 2013 Mar 26.
Article in English | MEDLINE | ID: mdl-23457265

ABSTRACT

Mutations in the Tar DNA binding protein of 43 kDa (TDP-43; TARDBP) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43(+) inclusions (FTLD-TDP). To determine the physiological function of TDP-43, we knocked out zebrafish Tardbp and its paralogue Tardbp (TAR DNA binding protein-like), which lacks the glycine-rich domain where ALS- and FTLD-TDP-associated mutations cluster. tardbp mutants show no phenotype, a result of compensation by a unique splice variant of tardbpl that additionally contains a C-terminal elongation highly homologous to the glycine-rich domain of tardbp. Double-homozygous mutants of tardbp and tardbpl show muscle degeneration, strongly reduced blood circulation, mispatterning of vessels, impaired spinal motor neuron axon outgrowth, and early death. In double mutants the muscle-specific actin binding protein Filamin Ca is up-regulated. Strikingly, Filamin C is similarly increased in the frontal cortex of FTLD-TDP patients, suggesting aberrant expression in smooth muscle cells and TDP-43 loss-of-function as one underlying disease mechanism.


Subject(s)
Axons/metabolism , DNA-Binding Proteins , Motor Neurons/metabolism , Muscular Atrophy/metabolism , Mutation , Vascular Diseases/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Axons/pathology , Contractile Proteins/genetics , Contractile Proteins/metabolism , Filamins , Humans , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Motor Neurons/pathology , Muscular Atrophy/genetics , Muscular Atrophy/pathology , Protein Structure, Tertiary , Vascular Diseases/genetics , Vascular Diseases/pathology , Zebrafish Proteins/genetics
8.
J Neurochem ; 127(4): 471-81, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23406323

ABSTRACT

Alzheimer's disease is the most frequent dementia. Pathologically, Alzheimer's disease is characterized by the accumulation of senile plaques composed of amyloid ß-peptide (Aß). Two proteases, ß- and γ-secretase proteolytically generate Aß from its precursor, the ß-amyloid precursor protein (APP). Inhibition of ß-secretase, also referred to as beta-site APP cleaving enzyme (BACE1) or γ-secretase is therefore of prime interest for the development of amyloid-lowering drugs. To assess the in vivo function of zebrafish Bace1 (zBace1), we generated zBace1 knock out fish by zinc finger nuclease-mediated genome editing. bace1 mutants (bace1-/-) are hypomyelinated in the PNS while the CNS is not affected. Moreover, the number of mechanosensory neuromasts is elevated in bace1-/-. Mutations in zebrafish Bace2 (zBace2) revealed a distinct melanocyte migration phenotype, which is not observed in bace1-/-. Double homozygous bace1-/-; bace2-/- fish do not enhance the single mutant phenotypes indicating non-redundant distinct physiological functions. Single homozygous bace1 mutants as well as double homozygous bace1 and bace2 mutants are viable and fertile suggesting that Bace1 is a promising drug target without major side effects. The identification of a specific bace2 -/- associated phenotype further allows improving selective Bace1 inhibitors and to distinguish between Bace 1 and Bace 2 inhibition in vivo. Inhibition of BACE1 protease activity has therapeutic importance for Alzheimer's disease. Analysis of BACE1 and BACE2 knock-out zebrafish revealed that they exhibit distinct phenotypes. bace1 mutants display hypomyelination in the PNS and supernumerary neuromasts while in bace2 mutants the shape and migration of melanocytes is affected. These phenotypes are not further enhanced in the viable double mutants. Our data suggest that blocking BACE1 activity is a safe therapeutic approach.


Subject(s)
Amyloid Precursor Protein Secretases/genetics , Melanocytes/physiology , Zebrafish Proteins/genetics , Zebrafish/metabolism , Amyloid Precursor Protein Secretases/metabolism , Animals , Animals, Genetically Modified , Cell Movement , Gene Knockout Techniques , Larva/genetics , Larva/metabolism , Models, Animal , Mutation , Phenotype , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish Proteins/metabolism
9.
J Neurosci ; 32(46): 16203-12, 2012 Nov 14.
Article in English | MEDLINE | ID: mdl-23152604

ABSTRACT

Mitochondria provide ATP, maintain calcium homeostasis, and regulate apoptosis. Neurons, due to their size and complex geometry, are particularly dependent on the proper functioning and distribution of mitochondria. Thus disruptions of these organelles and their transport play a central role in a broad range of neurodegenerative diseases. While in vitro studies have greatly expanded our knowledge of mitochondrial dynamics, our understanding in vivo remains limited. To address this shortcoming, we developed tools to study mitochondrial dynamics in vivo in optically accessible zebrafish. We demonstrate here that our newly generated tools, including transgenic "MitoFish," can be used to study the in vivo "life cycle" of mitochondria and allows identifying pharmacological and genetic modulators of mitochondrial dynamics. Furthermore we observed profound mitochondrial transport deficits in real time in a zebrafish tauopathy model. By rescuing this phenotype using MARK2 (microtubule-affinity regulating kinase 2), we provide direct in vivo evidence that this kinase regulates axonal transport in a Tau-dependent manner. Thus, our approach allows detailed studies of the dynamics of mitochondria in their natural environment under normal and disease conditions.


Subject(s)
Mitochondria/pathology , Nervous System Diseases/pathology , Zebrafish/physiology , Animals , Animals, Genetically Modified , Antineoplastic Agents/pharmacology , Biological Transport/physiology , Blotting, Western , Image Processing, Computer-Assisted , Mitochondria/ultrastructure , Nocodazole/pharmacology , Phenotype , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Sensory Receptor Cells/physiology , Sensory Receptor Cells/ultrastructure , tau Proteins/genetics
10.
EMBO J ; 29(16): 2841-57, 2010 Aug 18.
Article in English | MEDLINE | ID: mdl-20606625

ABSTRACT

Mutations in fused in sarcoma (FUS) are a cause of familial amyotrophic lateral sclerosis (fALS). Patients carrying point mutations in the C-terminus of FUS show neuronal cytoplasmic FUS-positive inclusions, whereas in healthy controls, FUS is predominantly nuclear. Cytoplasmic FUS inclusions have also been identified in a subset of frontotemporal lobar degeneration (FTLD-FUS). We show that a non-classical PY nuclear localization signal (NLS) in the C-terminus of FUS is necessary for nuclear import. The majority of fALS-associated mutations occur within the NLS and impair nuclear import to a degree that correlates with the age of disease onset. This presents the first case of disease-causing mutations within a PY-NLS. Nuclear import of FUS is dependent on Transportin, and interference with this transport pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules. Moreover, proteins known to be stress granule markers co-deposit with inclusions in fALS and FTLD-FUS patients, implicating stress granule formation in the pathogenesis of these diseases. We propose that two pathological hits, namely nuclear import defects and cellular stress, are involved in the pathogenesis of FUS-opathies.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Karyopherins/metabolism , Point Mutation , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolism , Active Transport, Cell Nucleus , Amino Acid Sequence , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Cells, Cultured , Cytoplasmic Granules/pathology , Gene Knockdown Techniques , HeLa Cells , Humans , Karyopherins/genetics , Molecular Sequence Data , Neurons/pathology , Protein Structure, Tertiary , RNA-Binding Protein FUS/analysis , RNA-Binding Protein FUS/chemistry , Zebrafish/embryology
11.
Neurobiol Dis ; 39(3): 265-71, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20381619

ABSTRACT

Methylene blue is an FDA approved compound with a variety of pharmacologic activities. It inhibits aggregation of several amyloidogenic proteins known to be deposited in neurodegenerative diseases. Recently, it has been proposed that methylene blue shows significant beneficial effects in a phase 2 clinical trial by slowing cognitive decline in Alzheimer's disease patients. To analyze its therapeutic potential, we investigated the effect of methylene blue on neurotoxicity in a zebrafish model for tauopathies. Transgenic expression of the frontotemporal dementia associated Tau-P301L mutation recapitulates a number of the pathological features observed in humans including abnormal phosphorylation and folding of Tau, tangle formation and Tau dependent neuronal loss. Upon incubation of zebrafish larvae with methylene blue, neither abnormal phosphorylation nor neuronal cell loss, reduced neurite outgrowth or a swimming defect were rescued. Methylene blue is biologically active in zebrafish since it reduced aggregation of a huntingtin variant containing a stretch of 102 glutamine residues. However, although huntingtin aggregation was largely prevented by methylene blue, huntingtin-dependent toxicity was unaffected. Our findings are consistent with the hypothesis that toxicity is not necessarily associated with deposition of insoluble amyloid proteins.


Subject(s)
Cell Death/drug effects , Methylene Blue/pharmacology , Neurons/drug effects , Peptides/metabolism , Tauopathies/metabolism , tau Proteins/metabolism , Animals , Animals, Genetically Modified/metabolism , Axons/drug effects , Blotting, Western , Fluorescent Antibody Technique , Neurons/metabolism , Peptides/genetics , Phosphorylation/drug effects , Tauopathies/genetics , Zebrafish/genetics , Zebrafish/metabolism , tau Proteins/genetics
12.
J Biol Chem ; 283(3): 1744-1753, 2008 Jan 18.
Article in English | MEDLINE | ID: mdl-17984093

ABSTRACT

Loss of function mutations in progranulin cause tau-negative frontotemporal lobar degeneration with ubiquitin-positive inclusions. A major protein component of these inclusions is TDP-43, which becomes hyperphosphorylated, ubiquitinated, and cleaved to generate C-terminal fragments, which apparently translocate from nuclei to the cytoplasm. Most progranulin mutations are nonsense mutations resulting in nonsense-mediated mRNA decay and consequently reduced progranulin protein levels. However, some missense mutations are described that occur within the signal sequence and mature progranulin. We now demonstrate that a progranulin mutation located within the signal sequence (PGRN A9D) results in cytoplasmic missorting with extremely low expression. In contrast, two other progranulin mutations (PGRN P248L and R432C) are expressed as immature proteins but are inefficiently transported through and partially degraded within the secretory pathway, resulting in a significantly reduced secretion. Thus apparently all progranulin mutations cause reduced protein expression or secretion, although by different cellular mechanisms. To investigate a putative relationship between reduced expression of progranulin and TDP-43 relocalization and deposition, we down-regulated progranulin in human cell lines and in zebrafish. Upon reduction of progranulin, neither a major redistribution of TDP-43 nor proteolytic processing to disease-characterizing C-terminal fragments could be observed.


Subject(s)
Dementia/genetics , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Intranuclear Inclusion Bodies/metabolism , Mutation, Missense/genetics , Ubiquitin/immunology , Zebrafish/metabolism , Amino Acid Sequence , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Down-Regulation , Gene Expression Regulation, Developmental , HeLa Cells , Humans , Intercellular Signaling Peptides and Proteins/chemistry , Molecular Sequence Data , Mutant Proteins/metabolism , Progranulins , Protein Processing, Post-Translational , Protein Transport , Subcellular Fractions/metabolism , Zebrafish/genetics , Zebrafish Proteins/metabolism
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