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 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 Mitochondrial damage by α-synuclein causes cell death in human dopaminergic neurons

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Goutham K. Ganjam ◽  
Kathrin Bolte ◽  
Lina A. Matschke ◽  
Sandra Neitemeier ◽  
Amalia M. Dolga ◽  
...  
Keyword(s):  
Cell Death ◽  
Dopaminergic Neurons ◽  
Fluorescent Protein ◽  
Network Activity ◽  
Mitochondrial Morphology ◽  
Therapeutic Effects ◽  
Caspase 1 ◽  
Neuronal Network Activity ◽  
Compound Screening ◽  
And Function

AbstractEvolving concepts on Parkinson’s disease (PD) pathology suggest that α-synuclein (aSYN) promote dopaminergic neuron dysfunction and death through accumulating in the mitochondria. However, the consequence of mitochondrial aSYN localisation on mitochondrial structure and bioenergetic functions in neuronal cells are poorly understood. Therefore, we investigated deleterious effects of mitochondria-targeted aSYN in differentiated human dopaminergic neurons in comparison with wild-type (WT) aSYN overexpression and corresponding EGFP (enhanced green fluorescent protein)-expressing controls. Mitochondria-targeted aSYN enhanced mitochondrial reactive oxygen species (ROS) formation, reduced ATP levels and showed severely disrupted structure and function of the dendritic neural network, preceding neuronal death. Transmission electron microscopy illustrated distorted cristae and many fragmented mitochondria in response to WT-aSYN overexpression, and a complete loss of cristae structure and massively swollen mitochondria in neurons expressing mitochondria-targeted aSYN. Further, the analysis of mitochondrial bioenergetics in differentiated dopaminergic neurons, expressing WT or mitochondria-targeted aSYN, elicited a pronounced impairment of mitochondrial respiration. In a pharmacological compound screening, we found that the pan-caspase inhibitors QVD and zVAD-FMK, and a specific caspase-1 inhibitor significantly prevented aSYN-induced cell death. In addition, the caspase inhibitor QVD preserved mitochondrial function and neuronal network activity in the human dopaminergic neurons overexpressing aSYN. Overall, our findings indicated therapeutic effects by caspase-1 inhibition despite aSYN-mediated alterations in mitochondrial morphology and function.

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 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.

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.

scholarly journals Mitochondrial dysfunction impairs human neuronal development and reduces neuronal network activity and synchronicity

2019 ◽  
Author(s):  
T.M. Klein Gunnewiek ◽  
E. J. H. Van Hugte ◽  
M. Frega ◽  
G. Solé Guardia ◽  
K.B. Foreman ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Mitochondrial Dysfunction ◽  
Cortical Neurons ◽  
Nuclear Dna ◽  
Induced Pluripotent Stem Cell ◽  
Mitochondrial Diseases ◽  
Network Activity ◽  
Neural Structure ◽  
Neuronal Network Activity

SummaryEpilepsy, intellectual and cortical sensory deficits and psychiatric manifestations are among the most frequent manifestations of mitochondrial diseases. Yet, how mitochondrial dysfunction affects neural structure and function remains largely elusive. This is mostly due to the lack of a proper in vitro translational neuronal model system(s) with impaired energy metabolism. Leveraging the induced pluripotent stem cell technology, from a cohort of patients with the common pathogenic m.3243A>G variant of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), we differentiated excitatory cortical neurons (iNeurons) with normal (low heteroplasmy) and impaired (high heteroplasmy) mitochondrial function on an isogenic nuclear DNA background. iNeurons with high levels of heteroplasmy exhibited mitochondrial dysfunction, delayed neural maturation, reduced dendritic complexity and fewer functional excitatory synapses. Micro-electrode array recordings of neuronal networks with high heteroplasmy displayed reduced network activity and decreased synchronous network bursting. The impaired neural energy metabolism of iNeurons compromising the structural and functional integrity of neurons and neural networks, could be the primary driver of increased susceptibility to neuropsychiatric manifestations of mitochondrial disease.

scholarly journals Applicability of hiPSC-Derived Neuronal Cocultures and Rodent Primary Cortical Cultures for In Vitro Seizure Liability Assessment

2020 ◽  
Vol 178 (1) ◽  
pp. 71-87
Author(s):  
Anke M Tukker ◽  
Fiona M J Wijnolts ◽  
Aart de Groot ◽  
Remco H S Westerink
Keyword(s):  
Microelectrode Array ◽  
Activity Patterns ◽  
Primary Cultures ◽  
Induced Pluripotent Stem Cell ◽  
Network Activity ◽  
Neuronal Network Activity ◽  
Cortical Cultures ◽  
Life Threatening ◽  
Induced Pluripotent

Abstract Seizures are life-threatening adverse drug reactions which are investigated late in drug development using rodent models. Consequently, if seizures are detected, a lot of time, money and animals have been used. Thus, there is a need for in vitro screening models using human cells to circumvent interspecies translation. We assessed the suitability of cocultures of human-induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes compared with rodent primary cortical cultures for in vitro seizure liability assessment using microelectrode arrays. hiPSC-derived and rodent primary cortical neuronal cocultures were exposed to 9 known (non)seizurogenic compounds (pentylenetetrazole, amoxapine, enoxacin, amoxicillin, linopirdine, pilocarpine, chlorpromazine, phenytoin, and acetaminophen) to assess effects on neuronal network activity using microelectrode array recordings. All compounds affect activity in hiPSC-derived cocultures. In rodent primary cultures all compounds, except amoxicillin changed activity. Changes in activity patterns for both cell models differ for different classes of compounds. Both models had a comparable sensitivity for exposure to amoxapine (lowest observed effect concentration [LOEC] 0.03 µM), linopirdine (LOEC 1 µM), and pilocarpine (LOEC 0.3 µM). However, hiPSC-derived cultures were about 3 times more sensitive for exposure to pentylenetetrazole (LOEC 30 µM) than rodent primary cortical cultures (LOEC 100 µM). Sensitivity of hiPSC-derived cultures for chlorpromazine, phenytoin, and enoxacin was 10-30 times higher (LOECs 0.1, 0.3, and 0.1 µM, respectively) than in rodent cultures (LOECs 10, 3, and 3 µM, respectively). Our data indicate that hiPSC-derived neuronal cocultures may outperform rodent primary cortical cultures with respect to detecting seizures, thereby paving the way towards animal-free seizure assessment.

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