scholarly journals Impaired M-current in KCNQ2 Encephalopathy Evokes Dyshomeostatic Modulation of Excitability

2019 ◽  
Author(s):  
Dina Simkin ◽  
Timothy J. Searl ◽  
Brandon N. Piyevsky ◽  
Marc Forrest ◽  
Luis A. Williams ◽  
...  
Keyword(s):  
Disease Model ◽  
Epileptic Encephalopathy ◽  
Bk Channels ◽  
Disease Stage ◽  
Loss Of Function ◽  
Therapeutic Implications ◽  
M Current ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
And Function

ABSTRACTMutations in KCNQ2, which encodes a pore-forming K+ channel subunit responsible for neuronal M-current, cause neonatal epileptic encephalopathy, a complex disorder presenting with severe early-onset seizures and impaired neurodevelopment. The condition is exceptionally difficult to treat, partially because the effects of KCNQ2 mutations on the development and function of human neurons are unknown. Here, we used induced pluripotent stem cells and gene editing to establish a disease model, and measured the functional properties of patient-derived neurons using electrophysiological and optical approaches. We find that while patient-derived excitatory neurons exhibit reduced M-current early, they develop intrinsic and network hyperexcitability progressively. This hyperexcitability is associated with faster action potential repolarization, larger afterhyperpolarization, and a functional enhancement of large conductance Ca2+-activated K+ (BK) channels. These properties facilitate a burst-suppression firing pattern that is reminiscent of the interictal electroencephalography pattern in patients. Importantly, we were able to phenocopy these excitability features in control neurons only by chronic but not acute pharmacological inhibition of M-current. Our findings suggest that dyshomeostatic mechanisms compound KCNQ2 loss-of-function and lead to alterations in the neurodevelopmental trajectory of patient-derived neurons. Our work has therapeutic implications in explaining why KCNQ2 agonists are not beneficial unless started at an early disease stage.

scholarly journals Dyshomeostatic modulation of Ca2+-activated K+ channels in a human neuronal model of KCNQ2 encephalopathy

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Dina Simkin ◽  
Kelly A Marshall ◽  
Carlos G Vanoye ◽  
Reshma R Desai ◽  
Bernabe I Bustos ◽  
...  
Keyword(s):  
Disease Model ◽  
Neuronal Model ◽  
Epileptic Encephalopathy ◽  
Loss Of Function ◽  
M Current ◽  
Complex Disorder ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
And Function ◽  
Action Potential Repolarization

Mutations in KCNQ2, which encodes a pore-forming K+ channel subunit responsible for neuronal M-current, cause neonatal epileptic encephalopathy, a complex disorder presenting with severe early-onset seizures and impaired neurodevelopment. The condition is exceptionally difficult to treat, partially because the effects of KCNQ2 mutations on the development and function of human neurons are unknown. Here, we used induced pluripotent stem cells (iPSCs) and gene editing to establish a disease model and measured the functional properties of differentiated excitatory neurons. We find that patient iPSC-derived neurons exhibit faster action potential repolarization, larger post-burst afterhyperpolarization and a functional enhancement of Ca2+-activated K+ channels. These properties, which can be recapitulated by chronic inhibition of M-current in control neurons, facilitate a burst-suppression firing pattern that is reminiscent of the interictal electroencephalography pattern in patients. Our findings suggest that dyshomeostatic mechanisms compound KCNQ2 loss-of-function leading to alterations in the neurodevelopmental trajectory of patient iPSC-derived neurons.

scholarly journals Fragile X syndrome patient-derived neurons developing in the mouse brain show FMR1 -dependent phenotypes

2021 ◽  
Author(s):  
Marine A Krzisch ◽  
Hao A Wu ◽  
Bingbing Yuan ◽  
Troy W. Whitfield ◽  
X. Shawn Liu ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Neuronal Development ◽  
Fragile X ◽  
In Vitro Models ◽  
Synaptic Activity ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
And Function ◽  
The Brain

Abnormal neuronal development in Fragile X syndrome (FXS) is poorly understood. Data on FXS patients remain scarce and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. Here, we co-injected neural precursor cells (NPCs) from FXS patient-derived and corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Single-cell RNA sequencing of transplanted cells revealed upregulated excitatory synaptic transmission and neuronal differentiation pathways in FXS neurons. Immunofluorescence analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, increased percentages of Arc- and Egr1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons pointed to an increase in synaptic activity and synaptic strength as compared to control. This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3D context, and could be used to test new therapeutic compounds correcting neuronal development defects in FXS.

scholarly journals High-Throughput Phenotypic Assay for Compounds That Influence Mitochondrial Health Using iPSC-Derived Human Neurons

2021 ◽  
pp. 247255522110006
Author(s):  
Courtney MacMullen ◽  
Ronald L. Davis
Keyword(s):  
Neurodegenerative Diseases ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mitochondrial Dynamics ◽  
Live Cell ◽  
Phenotypic Assay ◽  
Microtiter Plates ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
And Function

There is a critical need to develop high-throughput assays to identify compounds that offer therapy for individuals suffering from neurodegenerative diseases. Most brain disorders, including neurodegenerative diseases, share the common neuropathology of mitochondria dysfunction, which can lead to apoptosis of neurons, overproduction of reactive oxygen species (ROS), and other cellular neuropathologies characteristic of these diseases. Human induced pluripotent stem cells (iPSCs) with a stable genomic insertion of the neurogenin-2 transcription factor under the control of the TetOn promoter can be differentiated into excitatory human neurons (i3Neurons) within 3 days of exposure to doxycycline. These neurons have been used to develop and validate a live-cell assay for parameters of mitochondrial dynamics and function using two compounds known to promote mitochondrial elongation in mouse neurons, 4-hydroxychalcone and 2,4-dihyrdroxychalcone. The assay involves plating the neurons in 384-well microtiter plates, treating them with known or unknown substances, and then capturing morphological information for the neuronal mitochondria using a lentivirus vector to express a mitochondrial-targeted fluorescence reporter. The i3Neuron cultures exposed to these two compounds for 24 h exhibit significantly decreased circularity and significantly increased length compared to controls, two morphological parameters correlated with increased mitochondrial health. The assay is rapid, with results obtained after a one-week-long i3Neuron culture or one month if neurons are co-cultured with astrocytes. This live-cell, mitochondrial phenotypic assay can be used for high-throughput screening or as an orthogonal assay for compounds obtained via other high-throughput screening campaigns.

scholarly journals Lysosomal perturbations in dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation

2019 ◽  
Author(s):  
Justyna Okarmus ◽  
Helle Bogetofte ◽  
Sissel Ida Schmidt ◽  
Matias Ryding ◽  
Silvia Garcia Lopez ◽  
...  
Keyword(s):  
Dopaminergic Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Autophagic Flux ◽  
Compensatory Mechanism ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Tem Analysis ◽  
Induced Pluripotent ◽  
And Function ◽  
The Impact

AbstractMutations in the PARK2 gene encoding parkin, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson’s disease (PD). While parkin has been implicated in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration in both sporadic and familial PD upon parkin loss-of-function mutations remains unknown. Cultures of isogenic induced pluripotent stem cell (iPSC) lines with and without PARK2 knockout (KO) enable mechanistic studies of the effect of parkin deficiency in human dopaminergic neurons. In the present study, we used such cells to investigate the impact of PARK2 KO on the lysosomal compartment combining different approaches, such as mass spectrometry-based proteomics, electron microscopy (TEM) analysis and functional assays. We discovered a clear link between parkin deficiency and lysosomal alterations. PARK2 KO neurons exhibited a perturbed lysosomal morphology, displaying significantly enlarged and electron-lucent lysosomes as well as an increased total lysosomal content, which was exacerbated by mitochondrial stress. In addition, we found perturbed autophagic flux and decreased lysosomal enzyme activity suggesting an impairment of the autophagy-lysosomal pathway in parkin-deficient cells. Interestingly, activity of the GBA-encoded enzyme, β-glucocerebrosidase, was significantly increased suggesting the existence of a compensatory mechanism. In conclusion, our data provide a unique characterization of the morphology, content, and function of lysosomes in PARK2 KO neurons, thus revealing a new important connection between mitochondrial dysfunction and lysosomal dysregulation in PD pathogenesis.

scholarly journals Targeting the Microtubule EB1-CLASP2 Complex Modulates Na V 1.5 at Intercalated Discs

2021 ◽  
Author(s):  
Gerard A Marchal ◽  
Mariam Jouni ◽  
David Y Chiang ◽  
Marta Pérez-Hernández Duran ◽  
Svitlana Podliesna ◽  
...  
Keyword(s):  
Sodium Current ◽  
Induced Pluripotent Stem Cell ◽  
Loss Of Function ◽  
Whole Cell ◽  
Intercalated Discs ◽  
Cardiac Sodium Channel ◽  
Optical Reconstruction ◽  
Induced Pluripotent ◽  
And Function

Rationale: Loss-of-function of the cardiac sodium channel Na V 1.5 causes conduction slowing and arrhythmias. Na V 1.5 is differentially distributed within subcellular domains of cardiomyocytes, with sodium current (I Na ) being enriched at the intercalated discs (ID). Various pathophysiological conditions associated with lethal arrhythmias display ID-specific I Na reduction, but the mechanisms underlying microdomain-specific targeting of Na V 1.5 remain largely unknown. Objective: To investigate the role of the microtubule (MT) plus-end tracking proteins end binding protein 1 (EB1) and CLIP-associated protein 2 (CLASP2) in mediating Na V 1.5 trafficking and subcellular distribution in cardiomyocytes. Methods and Results: EB1 overexpression in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) resulted in enhanced whole-cell I Na , increased action potential (AP) upstroke velocity (V max ), and enhanced Na V 1.5 localization at the plasma membrane as detected by multi-color stochastic optical reconstruction microscopy (STORM). Fluorescence recovery after photobleaching (FRAP) experiments in HEK293A cells demonstrated that EB1 overexpression promoted Na V 1.5 forward trafficking. Knockout of MAPRE1 in hiPSC-CMs led to reduced whole-cell I Na , decreased V max and AP duration (APD) prolongation. Similarly, acute knockout of the MAPRE1 homolog in zebrafish (mapre1b) resulted in decreased ventricular conduction velocity and V max as well as increased APD. STORM imaging and macropatch I Na measurements showed that subacute treatment (2-3 hours) with SB216763 (SB2), a GSK3β inhibitor known to modulate CLASP2-EB1 interaction, reduced GSK3β localization and increased Na V 1.5 and I Na preferentially at the ID region of wild type murine ventricular cardiomyocytes. By contrast, SB2 did not affect whole cell I Na or Na V 1.5 localization in cardiomyocytes from Clasp2-deficient mice, uncovering the crucial role of CLASP2 in SB2-mediated modulation of NaV1.5 at the ID. Conclusions: Our findings demonstrate the modulatory effect of the MT plus-end tracking protein EB1 on Na V 1.5 trafficking and function, and identify the EB1-CLASP2 complex as a target for preferential modulation of I Na within the ID region of cardiomyocytes.

scholarly journals Loss-of-function variants in the schizophrenia risk gene SETD1A alter neuronal network activity in human neurons through cAMP/PKA pathway

2021 ◽  
Author(s):  
Shan Wang ◽  
Jon-Ruben van Rhijn ◽  
Ibrahim A Akkouh ◽  
Naoki Kogo ◽  
Nadine Maas ◽  
...  
Keyword(s):  
Neuronal Network ◽  
Synaptic Function ◽  
Network Activity ◽  
Loss Of Function ◽  
Functional Changes ◽  
Neuronal Network Activity ◽  
Camp Pathway ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
Pka Pathway

Heterozygous loss-of-function (LoF) mutations in SETD1A, which encodes a subunit of histone H3 lysine 4 methyltransferase, have been shown to cause a novel neurodevelopmental syndrome and increase the risk for schizophrenia. To study the effect of decreased SETD1A function in human cells, we generated excitatory/inhibitory neuronal networks from human induced pluripotent stem cells with a SETD1A heterozygous LoF mutation (SETD1A+/-). Our data show that SETD1A haploinsufficiency resulted in altered neuronal network activity, which was mainly characterized by an overly synchronized network. In individual neurons, this network phenotype was reflected by increased somatodendritic complexity and elevated synaptic connectivity. We found that this network phenotype was driven by SETD1A haploinsufficiency in glutamatergic neurons. In accordance with the functional changes, transcriptomic profiling revealed perturbations in gene sets associated with schizophrenia, synaptic transmission and glutamatergic synaptic function. At the molecular level, we identified specific changes in the cAMP/PKA pathway pointing toward a hyperactive cAMP pathway in SETD1A+/- neurons. Finally, using pharmacological experiments targeting the cAMP pathway we were able to rescue the network deficits in SETD1A+/- cultures. In conclusion, our results illuminate key molecular, cellular and network abnormalities caused by SETD1A haploinsufficiency and demonstrate a direct link between SETD1A and the cAMP-dependent pathway in human neurons.

scholarly journals Loss-of-function of OTUD7A in the schizophrenia-associated 15q13.3 deletion impairs synapse development and function in human neurons

2022 ◽  
Author(s):  
Alena Kozlova ◽  
Siwei Zhang ◽  
Alex V. Kotlar ◽  
Brendan Jamison ◽  
Hanwen Zhang ◽  
...  
Keyword(s):  
Copy Number Variants ◽  
Induced Pluripotent Stem Cell ◽  
Synaptic Proteins ◽  
Network Activity ◽  
Synapse Development ◽  
Loss Of Function ◽  
Causative Gene ◽  
Neuronal Network Activity ◽  
Human Neurons ◽  
And Function

Identifying causative gene(s) within disease-associated large genomic regions of copy number variants (CNVs) is challenging. Here, by targeted sequencing of genes within schizophrenia (SZ)-associated CNVs in 1,779 SZ cases and 1,418 controls, we identified three rare putative loss-of-function (LoF) mutations in OTU deubiquitinase 7A (OTUD7A) within the 15q13.3 deletion in cases, but none in controls. To tie OTUD7A LoF with any SZ-relevant cellular phenotypes, we modeled the OTUD7A LoF mutation, rs757148409, in human induced pluripotent stem cell (hiPSC)-derived induced excitatory neurons (iNs) by CRISPR/Cas9 engineering. The mutant iNs showed a ~50% decrease in OTUD7A expression without undergoing nonsense-mediated mRNA decay. The mutant iNs also exhibited marked reduction of dendritic complexity, density of synaptic proteins GluA1 and PSD-95, and neuronal network activity. Congruent with the neuronal phenotypes in mutant iNs, our transcriptomic analysis showed that the set of OTUD7A LoF-downregulated genes was enriched for those relating to synapse development and function, and was associated with SZ and other neuropsychiatric disorders. These results suggest that OTUD7A LoF impairs synapse development and neuronal function in human neurons, providing mechanistic insight into the possible role of OTUD7A in driving neuropsychiatric phenotypes associated with the 15q13.3 deletion.

scholarly journals PAX1 is essential for development and function of the human thymus

2020 ◽  
Vol 5 (44) ◽  
pp. eaax1036 ◽  
Author(s):  
Yasuhiro Yamazaki ◽  
Raul Urrutia ◽  
Luis M. Franco ◽  
Silvia Giliani ◽  
Kejian Zhang ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Rare Variants ◽  
Severe Combined Immunodeficiency ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Thymus Development ◽  
Induced Pluripotent ◽  
And Function ◽  
Epithelial Progenitor Cells

We investigated the molecular and cellular basis of severe combined immunodeficiency (SCID) in six patients with otofaciocervical syndrome type 2 who failed to attain T cell reconstitution after allogeneic hematopoietic stem cell transplantation, despite successful engraftment in three of them. We identified rare biallelic PAX1 rare variants in all patients. We demonstrated that these mutant PAX1 proteins have an altered conformation and flexibility of the paired box domain and reduced transcriptional activity. We generated patient-derived induced pluripotent stem cells and differentiated them into thymic epithelial progenitor cells and found that they have an altered transcriptional profile, including for genes involved in the development of the thymus and other tissues derived from pharyngeal pouches. These results identify biallelic, loss-of-function PAX1 mutations as the cause of a syndromic form of SCID due to altered thymus development.

Nuclear accumulation of CHMP7 initiates nuclear pore complex injury and subsequent TDP-43 dysfunction in sporadic and familial ALS

2021 ◽  
Vol 13 (604) ◽  
pp. eabe1923
Author(s):  
Alyssa N. Coyne ◽  
Victoria Baskerville ◽  
Benjamin L. Zaepfel ◽  
Dennis W. Dickson ◽  
Frank Rigo ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Induced Pluripotent Stem Cell ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Human Motor Cortex ◽  
Sporadic Als ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
And Function

Alterations in the components [nucleoporins (Nups)] and function of the nuclear pore complex (NPC) have been implicated as contributors to the pathogenesis of genetic forms of neurodegeneration including C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). We hypothesized that Nup alterations and the consequential loss of NPC function may lie upstream of TDP-43 dysfunction and mislocalization widely observed in ALS, FTD, and related neurodegenerative diseases. Here, we provide evidence that CHMP7, a critical mediator of NPC quality control, is increased in nuclei of C9orf72 and sporadic ALS induced pluripotent stem cell (iPSC)–derived spinal neurons (iPSNs) and postmortem human motor cortex before the emergence of Nup alterations. Inhibiting the nuclear export of CHMP7 triggered Nup reduction and TDP-43 dysfunction and pathology in human neurons. Knockdown of CHMP7 alleviated disease-associated Nup alterations, deficits in Ran GTPase localization, defects in TDP-43–associated mRNA expression, and downstream glutamate-induced neuronal death. Thus, our data support a role for altered CHMP7-mediated Nup homeostasis as a prominent initiating pathological mechanism for familial and sporadic ALS and highlight the potential for CHMP7 as therapeutic target.

The Spectrum of KCNQ2- and KCNQ3-Related Epilepsy

2021 ◽  
Author(s):  
Anna Portale ◽  
Mattia Comella ◽  
Giulia Salomone ◽  
Alessandra Di Nora ◽  
Lidia Marino ◽  
...  
Keyword(s):  
De Novo ◽  
Epileptic Encephalopathy ◽  
Response To Treatment ◽  
Psychomotor Development ◽  
K Channel ◽  
Loss Of Function ◽  
M Current ◽  
Wide Range ◽  
Cortical Visual Impairment ◽  
Infantile Epilepsy

Abstract KCNQ genes encode for a family of six transmembrane domains, single pore-loop, and K+ channel α-subunits that have a wide range of physiological correlates. In the brain, KCNQ2 and KCNQ3 heteromultimers are thought to underlie the M-current which is essential in raising the threshold for firing an action potential; mutations in these genes may cause several types of infantile epilepsies. KCNQ2-related disorders represent a continuum of overlapping neonatal epileptic phenotypes that range from KCNQ2 benign familial neonatal epilepsy (BFNE), a seizure disorder that occur in children who typically have a normal psychomotor development and are inherited as an autosomal dominant trait, to KCNQ2 early-onset epileptic encephalopathy (EOEE) as the result of a de novo pathogenic variant. KCNQ3-related disorders are rarer and include BFNE, benign familial infantile epilepsy and KCNQ3-related epileptic encephalopathy with intellectual disability with or without seizures and/or cortical visual impairment. For both KCNQ2- and KCNQ3-related disorders, it is possible to use several drugs for different classes of mutations (i.e., gain of function vs. loss of function), and usually their effects vary in relation to the clinical presentation and the phenotype of the patient. However, KCNQ2-EOEE patients have a worse response to treatment than KCNQ2-BFNE patients and usually become drug resistant with multiple daily seizures.

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