scholarly journals A Versatile Clustered Regularly Interspaced Palindromic Repeats Toolbox to Study Neurological CaV3.2 Channelopathies by Promoter-Mediated Transcription Control

2022 ◽  
Vol 14 ◽  
Author(s):  
Despina Tsortouktzidis ◽  
Anna R. Tröscher ◽  
Herbert Schulz ◽  
Thoralf Opitz ◽  
Susanne Schoch ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Target Genes ◽  
Low Voltage ◽  
Neurological Diseases ◽  
Epileptic Seizures ◽  
Loss Of Function ◽  
Transcription Control ◽  
Protein Variant ◽  
Knock Out ◽  
Endogenous Loci

Precise genome editing in combination with viral delivery systems provides a valuable tool for neuroscience research. Traditionally, the role of genes in neuronal circuits has been addressed by overexpression or knock-out/knock-down systems. However, those techniques do not manipulate the endogenous loci and therefore have limitations. Those constraints include that many genes exhibit extensive alternative splicing, which can be regulated by neuronal activity. This complexity cannot be easily reproduced by overexpression of one protein variant. The CRISPR activation and interference/inhibition systems (CRISPRa/i) directed to promoter sequences can modulate the expression of selected target genes in a highly specific manner. This strategy could be particularly useful for the overexpression of large proteins and for alternatively spliced genes, e.g., for studying large ion channels known to be affected in ion channelopathies in a variety of neurological diseases. Here, we demonstrate the feasibility of a newly developed CRISPRa/i toolbox to manipulate the promoter activity of the Cacna1h gene. Impaired, function of the low-voltage-activated T-Type calcium channel CaV3.2 is involved in genetic/mutational as well as acquired/transcriptional channelopathies that emerge with epileptic seizures. We show CRISPR-induced activation and inhibition of the Cacna1h locus in NS20Y cells and primary cortical neurons, as well as activation in mouse organotypic slice cultures. In future applications, the system offers the intriguing perspective to study functional effects of gain-of-function or loss-of-function variations in the Cacna1h gene in more detail. A better understanding of CaV3.2 channelopathies might result in a major advancement in the pharmacotherapy of CaV3.2 channelopathy diseases.

scholarly journals A versatile CRISPR toolbox to study neurological CaV3.2 channelopathies by promoter-mediated transcription control

2021 ◽  
Author(s):  
Despina Tsortouktzidis ◽  
Anna R. Tröscher ◽  
Herbert Schulz ◽  
Susanne Schoch ◽  
Albert J. Becker ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Target Genes ◽  
Low Voltage ◽  
Neurological Diseases ◽  
Epileptic Seizures ◽  
Loss Of Function ◽  
Transcription Control ◽  
Protein Variant ◽  
Knock Out ◽  
Endogenous Loci

AbstractPrecise genome editing in combination with viral delivery systems provides a valuable tool for neuroscience research. Traditionally, the role of genes in neuronal circuits has been addressed by overexpression or knock-out/knock-down systems. However, those techniques do not manipulate the endogenous loci and therefore have limitations. Those constraints include that many genes exhibit extensive alternative splicing, which can be regulated by neuronal activity. This complexity cannot be easily reproduced by overexpression of one protein variant. The CRISPR activation and interference/inhibition systems (CRISPRa/i) directed to promoter sequences can modulate the expression of selected target genes in a highly specific manner. This strategy could be particularly useful for the overexpression of large proteins and for alternatively spliced genes, e.g. for studying large ion channels known to be affected in ion channelopathies in a variety of neurological diseases. Here, we demonstrate the feasibility of a newly developed CRISPRa/i toolbox to manipulate the promoter activity of the Cacna1h gene. Impaired, function of the low-voltage-activated T-Type calcium channel CaV3.2 is involved in genetic/mutational as well as acquired/transcriptional channelopathies that emerge with epileptic seizures. We show CRISPR-induced activation and inhibition of the Cacna1h locus in NS20Y cells and primary cortical neurons, as well as activation in mouse organotypic slice cultures. In future applications, the system offers the intriguing perspective to study functional effects of gain-of-function or loss-of-function variations in the Cacna1h gene in more detail. A better understanding of CaV3.2 channelopathies might result in a major advancement in the pharmacotherapy of CaV3.2 channelopathy diseases.

scholarly journals Novel pre-clinical model for CDKL5 Deficiency Disorder

2021 ◽  
Author(s):  
Rita J Serrano ◽  
Clara Lee ◽  
Robert J Bryson-Richardson ◽  
Tamar Sztal
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Genetic Manipulation ◽  
Muscle Tone ◽  
Neurological Diseases ◽  
Epileptic Seizures ◽  
Loss Of Function ◽  
Zebrafish Model ◽  
Clinical Model

Cyclin-dependent kinase-like-5 (CDKL5) Deficiency Disorder (CDD) is a severe X-linked neurodegenerative disease characterized by early-onset epileptic seizures, low muscle tone, progressive intellectual disability, severe motor function and visual impairment. CDD affects approximately 1 in 60,000 live births with many patients dying by early adulthood. For many patients, quality of life is significantly reduced due to the severity of their neurological symptoms and functional impairment. There are no effective therapies for CDD with current treatments focusing on improving symptoms rather than addressing the underlying causes of the disorder. Zebrafish offer a number of unique advantages for high-throughput pre-clinical evaluation of potential therapies for human neurological diseases including CDD. In particular, the large number of zebrafish that can be produced, together with the possibilities for in vivo imaging and genetic manipulation, allows for the detailed assessment of disease pathogenesis and therapeutic discovery. We have characterised a loss of function zebrafish model for CDD, containing a nonsense mutation in cdkl5. cdkl5 mutant zebrafish display defects in neuronal patterning, microcephaly, and reduced muscle function caused by impaired muscle innervation. This study provides a powerful vertebrate model to investigate CDD disease pathophysiology and allow high-throughput screening for effective therapies.

scholarly journals Novel pre-clinical model for CDKL5 Deficiency Disorder

2021 ◽  
Author(s):  
Rita J. Serrano ◽  
Clara Lee ◽  
Alon M. Douek ◽  
Jan Kaslin ◽  
Robert J. Bryson-Richardson ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Genetic Manipulation ◽  
Muscle Tone ◽  
Neurological Diseases ◽  
Epileptic Seizures ◽  
Loss Of Function ◽  
Zebrafish Model ◽  
Clinical Model

Cyclin-Dependent Kinase-Like-5 (CDKL5) Deficiency Disorder (CDD) is a severe X-linked neurodegenerative disease characterized by early-onset epileptic seizures, low muscle tone, progressive intellectual disability, and severe motor function. CDD affects approximately 1 in 60,000 live births with many patients experiencing a reduced quality of life due to the severity of their neurological symptoms and functional impairment. There are no effective therapies for CDD with current treatments focusing on improving symptoms rather than addressing the underlying causes of the disorder. Zebrafish offer many unique advantages for high-throughput pre-clinical evaluation of potential therapies for neurological diseases, including CDD. In particular, the large number of offspring produced, together with the possibilities for in vivo imaging and genetic manipulation, allows for the detailed assessment of disease pathogenesis and therapeutic discovery. We have characterised a loss of function zebrafish model for CDD, containing a nonsense mutation in cdkl5. cdkl5 mutant zebrafish display defects in neuronal patterning, seizures, microcephaly, and reduced muscle function caused by impaired muscle innervation. This study provides a powerful vertebrate model to investigate CDD disease pathophysiology and allow high-throughput screening for effective therapies.

scholarly journals The rice pentatricopeptide repeat protein PPR756 is involved in pollen development by affecting multiple RNA editing in mitochondria

2019 ◽  
Author(s):  
Qiannan Zhang ◽  
Yanghong Xu ◽  
Jishuai Huang ◽  
Kai Zhang ◽  
Haijun Xiao ◽  
...  
Keyword(s):  
Rna Editing ◽  
Pollen Development ◽  
Target Genes ◽  
Large Family ◽  
Loss Of Function ◽  
Pentatricopeptide Repeat ◽  
Knock Out ◽  
Ppr Protein ◽  
Ppr Proteins ◽  
Sterile Pollen

AbstractIn land plants, the pentatricopeptide repeat (PPR) proteins form a large family involved in post-transcriptional processing of RNA in mitochondria and chloroplasts, which is critical for plant development and evolutionary adaption. Although studies showed a number of PPR proteins generally influence the editing of organellar genes, few of them were characterized in detail in rice. Here, we report a PLS-E subclass PPR protein in rice, PPR756, loss of function of which led to the abolishment of RNA editing events among three mitochondrial genes including atp6, ccmC, and nad7. Their defective C-to-U transformation then resulted in improper amino acid retention which could cause abortive pollen development. Furthermore, PPR756 could bind to the three target genes directly and interact with three OsMORFs (multiple organellar RNA editing factors): OsMORF1, OsMORF8-1, and OsMORF8-2. The knock-out plants of PPR756 exhibited retarded growth and greener leaves during the early vegetative stages, along with sterile pollen and lower seed setting at the reproductive stage. These results established a role for PPR756 in rice development, participating in RNA editing of three various transcripts and cooperating with OsMORFs via an editosome manner in rice.

scholarly journals The Molecular Function of PURA and Its Implications in Neurological Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Lena Molitor ◽  
Sabrina Bacher ◽  
Sandra Burczyk ◽  
Dierk Niessing
Keyword(s):  
Transcriptional Control ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Current Knowledge ◽  
Fragile X ◽  
Neurological Diseases ◽  
Neurodevelopmental Disorder ◽  
Epileptic Seizures ◽  
Knock Out ◽  
Dna And Rna

In recent years, genome-wide analyses of patients have resulted in the identification of a number of neurodevelopmental disorders. Several of them are caused by mutations in genes that encode for RNA-binding proteins. One of these genes is PURA, for which in 2014 mutations have been shown to cause the neurodevelopmental disorder PURA syndrome. Besides intellectual disability (ID), patients develop a variety of symptoms, including hypotonia, metabolic abnormalities as well as epileptic seizures. This review aims to provide a comprehensive assessment of research of the last 30 years on PURA and its recently discovered involvement in neuropathological abnormalities. Being a DNA- and RNA-binding protein, PURA has been implicated in transcriptional control as well as in cytoplasmic RNA localization. Molecular interactions are described and rated according to their validation state as physiological targets. This information will be put into perspective with available structural and biophysical insights on PURA’s molecular functions. Two different knock-out mouse models have been reported with partially contradicting observations. They are compared and put into context with cell biological observations and patient-derived information. In addition to PURA syndrome, the PURA protein has been found in pathological, RNA-containing foci of patients with the RNA-repeat expansion diseases such as fragile X-associated tremor ataxia syndrome (FXTAS) and amyotrophic lateral sclerosis (ALS)/fronto-temporal dementia (FTD) spectrum disorder. We discuss the potential role of PURA in these neurodegenerative disorders and existing evidence that PURA might act as a neuroprotective factor. In summary, this review aims at informing researchers as well as clinicians on our current knowledge of PURA’s molecular and cellular functions as well as its implications in very different neuronal disorders.

scholarly journals P.135 Autism-associated mutations in SHANK2 increase synaptic connectivity and dendrite complexity in human neurons

2018 ◽  
Vol 45 (s2) ◽  
pp. S52-S52
Author(s):  
K Zaslavsky ◽  
W Zhang ◽  
E Deneault ◽  
M Zhao ◽  
DC Rodrigues ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Autism Spectrum ◽  
Dermal Fibroblasts ◽  
Neuronal Morphology ◽  
Synaptic Connectivity ◽  
Loss Of Function ◽  
Knock Out ◽  
Electrophysiological Properties ◽  
Dendrite Length ◽  
Human Neurons

Background: Heterozygous loss-of-function mutations in the synaptic scaffolding gene SHANK2 are strongly associated with autism spectrum disorder (ASD). However, their impact on the function of human neurons is unknown. Derivation of induced pluripotent stem cells (iPSC) from affected individuals permits generation of live neurons to answer this question. Methods: We generated iPSCs by reprogramming dermal fibroblasts of neurotypic and ASD-affected donors. To isolate the effect of SHANK2, we used CRISPR/Cas9 to knock out SHANK2 in control iPSCs and correct a heterozygous nonsense mutation in ASD-affected donor iPSCs. We then derived cortical neurons from SOX1+ neural precursor cells differentiated from these iPSCs. Using a novel assay that overcomes line-to-line variability, we compared neuronal morphology, total synapse number, and electrophysiological properties between SHANK2 mutants and controls. Results: Relative to controls, SHANK2 mutant neurons have increased dendrite complexity, dendrite length, total synapse number (1.5-2-fold), and spontaneous excitatory postsynaptic current (sEPSC) frequency (3-7.6-fold). Conclusions: ASD-associated heterozygous loss-of-function mutations in SHANK2 increase synaptic connectivity among human neurons by increasing synapse number and sEPSC frequency. This is partially supported by increased dendrite length and complexity, providing evidence that SHANK2 functions as a suppressor of dendrite branching during neurodevelopment.

Treponema, Iron and Neurodegeneration

2018 ◽  
Vol 15 (8) ◽  
pp. 716-722 ◽  
Author(s):  
A. Jolivet-Gougeon ◽  
M. Bonnaure-Mallet
Keyword(s):  
Iron Metabolism ◽  
Plasma Proteins ◽  
Iron Homeostasis ◽  
Neurological Diseases ◽  
Host Cells ◽  
Loss Of Function ◽  
Binding Ability ◽  
Iron Binding Proteins ◽  
Oxidative Species ◽  
Iron Reductase

Spirochetes are suspected to be linked to the genesis of neurological diseases, including neurosyphillis or neurodegeneration (ND). Impaired iron homeostasis has been implicated in loss of function in several enzymes requiring iron as a cofactor, formation of toxic oxidative species, inflammation and elevated production of beta-amyloid proteins. This review proposes to discuss the link that may exist between the involvement of Treponema spp. in the genesis or worsening of ND, and iron dyshomeostasis. Proteins secreted by Treponema can act directly on iron metabolism, with hemin binding ability (HbpA and HbpB) and iron reductase able to reduce the central ferric iron of hemin, iron-containing proteins (rubredoxin, neelaredoxin, desulfoferrodoxin metalloproteins, bacterioferritins etc). Treponema can also interact with cellular compounds, especially plasma proteins involved in iron metabolism, contributing to the virulence of the syphilis spirochetes (e.g. treponemal motility and survival). Fibronectin, transferrin and lactoferrin were also shown to be receptors for treponemal adherence to host cells and extracellular matrix. Association between Treponema and iron binding proteins results in iron accumulation and sequestration by Treponema from host macromolecules during systemic and mucosal infections.

scholarly journals Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers

2020 ◽  
Vol 117 (41) ◽  
pp. 25486-25493 ◽  
Author(s):  
Jun Xu ◽  
Wei Wang ◽  
Liang Xu ◽  
Jia-Yu Chen ◽  
Jenny Chong ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Neurological Diseases ◽  
Transcription Elongation ◽  
Cockayne Syndrome ◽  
Stable Complex ◽  
Loss Of Function ◽  
Pol Ii ◽  
Chromatin Remodelers ◽  
Processivity Factor

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

scholarly journals Role of Satb1 and Satb2 Transcription Factors in the Glutamate Receptors Expression and Ca2+ Signaling in the Cortical Neurons In Vitro

2021 ◽  
Vol 22 (11) ◽  
pp. 5968
Author(s):  
Egor A. Turovsky ◽  
Maria V. Turovskaya ◽  
Evgeniya I. Fedotova ◽  
Alexey A. Babaev ◽  
Viktor S. Tarabykin ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nmda Receptor ◽  
Cortical Neurons ◽  
Target Genes ◽  
Cortical Cell ◽  
Inhibitory System ◽  
Selective Agonist ◽  
Genes Encoding ◽  
Deficient Mice

Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurodegenerative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, thereby leading to higher amplitudes of Ca2+-signals in neurons derived from the Satb1-deficient (Satb1fl/+ * NexCre/+) and Satb1-null mice (Satb1fl/fl * NexCre/+) in response to the selective agonist reducing the EC50 for the NMDA receptor. Simultaneously, there is an increase in the expression of the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca2+-signals of neurons in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory system of neurotransmission resulting in the suppression of the neuron maturation process and switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal network in the cortical cell culture.

scholarly journals Loss-of-function mutations in the histone methyltransferase EZH2 promote chemotherapy resistance in AML

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Julia M. Kempf ◽  
Sabrina Weser ◽  
Michael D. Bartoschek ◽  
Klaus H. Metzeler ◽  
Binje Vick ◽  
...  
Keyword(s):  
Target Genes ◽  
Chemotherapeutic Agent ◽  
Chemotherapy Resistance ◽  
Xenograft Model ◽  
Histone Methyltransferase ◽  
Loss Of Function ◽  
Resistance Development ◽  
Selective Growth Advantage ◽  
Patient Derived Xenograft ◽  
Transmembrane Transporter

AbstractChemotherapy resistance is the main impediment in the treatment of acute myeloid leukaemia (AML). Despite rapid advances, the various mechanisms inducing resistance development remain to be defined in detail. Here we report that loss-of-function mutations (LOF) in the histone methyltransferase EZH2 have the potential to confer resistance against the chemotherapeutic agent cytarabine. We identify seven distinct EZH2 mutations leading to loss of H3K27 trimethylation via multiple mechanisms. Analysis of matched diagnosis and relapse samples reveal a heterogenous regulation of EZH2 and a loss of EZH2 in 50% of patients. We confirm that loss of EZH2 induces resistance against cytarabine in the cell lines HEK293T and K562 as well as in a patient-derived xenograft model. Proteomics and transcriptomics analysis reveal that resistance is conferred by upregulation of multiple direct and indirect EZH2 target genes that are involved in apoptosis evasion, augmentation of proliferation and alteration of transmembrane transporter function. Our data indicate that loss of EZH2 results in upregulation of its target genes, providing the cell with a selective growth advantage, which mediates chemotherapy resistance.

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