Biology subjects

Schubert, D.

Publications and source records attributed to Schubert, D..

32 records · Page 2Linked to original sources

bioRxiv2020

Brunner syndrome associated MAOA dysfunction in human dopaminergic neurons results in NMDAR hyperfunction and increased network activity.

BackgroundMonoamine neurotransmitter abundance affects motor control, emotion, and cognitive function and is regulated by monoamine oxidases. Amongst these, monoamine oxidase A (MAOA) catalyzes the degradation of dopamine, norepinephrine, and serotonin into their inactive metabolites. Loss-of-function mutations in the X-linked MAOA gene cause Brunner syndrome, which is characterized by various forms of impulsivity, maladaptive externalizing behavior, and mild intellectual disability. Impaired MAOA activity in individuals with Brunner syndrome results in bioamine aberration, but it is currently unknown how this affects neuronal function. MethodsWe generated human induced pluripotent stem cell (hiPSC)-derived dopaminergic (DA) neurons from three individuals with Brunner syndrome carrying different mutations, and used CRISPR/Cas9 mediated homologous recombination to rescue MAOA function. We used these lines to characterize morphological and functional properties of DA neuronal cultures at the single cell and neuronal network level in vitro. ResultsBrunner syndrome DA neurons showed reduced synaptic density but hyperactive network activity. Intrinsic functional properties and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission were not affected by MAOA dysfunction. Instead, we show that the neuronal network hyperactivity is mediated by upregulation of the GRIN2A and GRIN2B subunits of the N-methyl-D-aspartate receptor (NMDAR), and rescue of MAOA results in normalization of NMDAR function as well as restoration of network activity. ConclusionsOur data suggest that MAOA dysfunction in Brunner syndrome increases activity of dopaminergic neurons through upregulation of NMDAR function, which may contribute to Brunner syndrome associated phenotypes.

neuroscience↗

bioRxiv2020

KANSL1 Deficiency Causes Neuronal Dysfunction by Oxidative Stress-Induced Autophagy

Autophagy is a finely tuned process of programmed degradation and recycling of proteins and cellular components, which is crucial in neuronal function and synaptic integrity. Mounting evidence implicates chromatin remodelling in fine-tuning autophagy pathways. However, this epigenetic regulation is poorly understood in neurons. Here, we investigate the role in autophagy of KANSL1, a member of the nonspecific lethal complex, which acetylates histone H4 on lysine 16 (H4K16ac) to facilitate transcriptional activation. Loss-of-function of KANSL1 is strongly associated with the neurodevelopmental disorder Koolen-de Vries Syndrome (KdVS). Starting from KANSL1-deficient human induced-pluripotent stem cells, both from KdVS patients and genome-edited lines, we identified superoxide dismutase 1, an antioxidant enzyme, to be significantly decreased, leading to a subsequent increase in oxidative stress and autophagosome accumulation. In KANSL1-deficient neurons, autophagosome accumulation at excitatory synapses resulted in reduced synaptic density, reduced AMPA receptor-mediated transmission and impaired neuronal network activity. Furthermore, we found that increased oxidative stress-mediated autophagosome accumulation leads to increased mTOR activation and decreased lysosome function, further preventing the clearing of autophagosomes. Finally, by pharmacologically reducing oxidative stress, we could rescue the aberrant autophagosome formation as well as synaptic and neuronal network activity in KANSL1-deficient neurons. Our findings thus point towards an important relation between oxidative stress-induced autophagy and synapse function, and demonstrate the importance of H4K16ac-mediated changes in chromatin structure to balance reactive oxygen species- and mTOR-dependent autophagy.

neuroscience↗

bioRxiv2020

Cadherin-13 is a critical regulator of GABAergic modulation in human stem cell derived neuronal networks

Activity in the healthy brain relies on concerted interplay of excitation (E) and inhibition (I) via balanced synaptic communication between glutamatergic and GABAergic neurons. A growing number of studies imply that disruption of this E/I balance is a commonality in many brain disorders, however, obtaining mechanistic insight into these disruptions, with translational value for the human patient, has typically been hampered by methodological limitations. Cadherin-13 (CDH13) has strongly been associated to attention-deficit/hyperactivity disorder and comorbid disorders such as autism and schizophrenia. CDH13 localises at inhibitory presynapses, specifically of parvalbumin (PV) and somatostatin (SST) expressing GABAergic neurons. However, the mechanism by which CDH13 regulates the function of inhibitory synapses in human neurons remains unknown. Starting from human induced pluripotent stem cells, we established a robust method to generate a homogenous population of SST and PV expressing GABAergic neurons (iGABA) in vitro, and co-cultured these with glutamatergic neurons at defined E/I ratios on micro-electrode arrays. We identified functional network parameters that are most reliably affected by GABAergic modulation as such, and through alterations of E/I balance by reduced expression of CDH13 in iGABAs. We found that CDH13-deficiency in iGABAs decreased E/I balance by means of increased inhibition. Moreover, CDH13 interacts with Integrin-{beta}1 and Integrin-{beta}3, which play opposite roles in the regulation of inhibitory synaptic strength via this interaction. Taken together, this model allows for standardized investigation of the E/I balance in a human neuronal background and can be deployed to dissect the cell-type specific contribution of disease genes to the E/I balance.

neuroscience↗

bioRxiv2020

Alternative splicing coupled nonsense-mediated decay shapes the temperature-dependent transcriptome

Mammalian body temperature oscillates with the time of the day and is altered in diverse pathological conditions. We recently identified a body temperature-sensitive thermometer-like kinase, which alters SR protein phosphorylation and thereby globally controls alternative splicing (AS). AS can generate mRNA variants containing premature termination codons, which are degraded by nonsense-mediated decay (NMD). Here we show extensive coupling of body temperature-controlled AS to NMD, leading to global control of temperature-dependent gene expression (GE). Temperature-controlled NMD-inducing splicing events are evolutionarily conserved and pervasively found within RNA-binding proteins, including most SR proteins. NMD-inducing exons are essential for rhythmic GE of SR proteins and have a global role in establishing temperature-dependent rhythmic GE profiles, both, in mammals under circadian body temperature cycles and in plants in response to ambient temperature changes. Together, these data identify body temperature-driven AS-NMD as an evolutionary ancient, core clock-independent mechanism to generate rhythmic GE.

molecular biology↗

bioRxiv2019

Characterization of SETD1A haploinsufficiency in humans and Drosophila defines a novel neurodevelopmental syndrome.

Defects in histone methyltransferases (HMTs) are major contributing factors in neurodevelopmental disorders (NDDs). Heterozygous variants of SETD1A involved in histone H3 lysine 4 (H3K4) methylation were previously identified in individuals with schizophrenia. Here, we define the clinical features of the Mendelian syndrome associated with haploinsufficiency of SETD1A by investigating 15 predominantly pediatric individuals who all have de novo SETD1A variants. These individuals present with a core set of symptoms comprising global developmental delay and/or intellectual disability, subtle facial dysmorphisms, behavioral and psychiatric problems. We examined cellular phenotypes in three patient derived lymphoblastoid cell lines with three variants: p.Gly535Alafs*12, c.4582-2_4582delAG, and p.Tyr1499Asp. These patient cell lines displayed DNA damage repair defects that were comparable to previously observed RNAi-mediated depletion of SETD1A. This suggested that these variants, including the p.Tyr1499Asp in the catalytic SET domain, behave as Loss-of-Function (LoF) alleles. Previous studies demonstrated a role for SETD1A in cell cycle control and differentiation. However, individuals with SETD1A variants do not show major structural brain defects or severe microcephaly, suggesting that defective proliferation and differentiation of neural progenitors is unlikely the single underlying cause of the disorder. We show here that the Drosophila Melanogaster SETD1A orthologue is required in postmitotic neurons of the fly brain for normal memory, suggesting a role in post development neuronal function. Together, this study defines a neurodevelopmental disorder caused by dominant de novo LoF variants in SETD1A and further supports a role for H3K4 methyltransferases in the regulation of neuronal processes underlying normal cognitive functioning.

genetics↗

bioRxiv2019

Serotonergic development of active sensing

Active sensing requires adaptive motor (positional) control of sensory organs based on contextual, sensory and task requirements, and develops postnatally after the maturation of intracortical circuits. Alterations in sensorimotor network connectivity during this period are likely to impact sensorimotor computation also in adulthood. Serotonin is among the cardinal developmental regulators of network formation, thus changing the serotonergic drive might have consequences for the emergence and maturation of sensorimotor control. Here we tested this hypothesis on an object localization task by quantifying the motor control dynamics of whiskers during tactile navigation. The results showed that sustained alterations in serotonergic signaling in serotonin transporter knockout rats, or the transient pharmacological inactivation of the transporter during early postnatal development, impairs the emergence of adaptive motor control of whisker position based on recent sensory information. A direct outcome of this altered motor control is that the mechanical force transmitted to whisker follicles upon contact is reduced, suggesting that increased excitability observed upon altered serotonergic signaling is not due to increased synaptic drive originating from the periphery upon whisker contact. These results argue that postnatal development of adaptive motor control requires intact serotonergic signaling and that even its transient dysregulation during early postnatal development causes lasting sensorimotor impairments in adulthood.

neuroscience↗

bioRxiv2019

Transcriptional and post-transcriptional regulation and transcriptional memory of chromatin regulators in response to low temperature

Chromatin regulation ensures stable repression of stress-inducible genes under non-stress conditions and transcriptional activation and memory of such an activation of those genes when plants are exposed to stress. However, there is only limited knowledge on how chromatin genes are regulated at the transcriptional and post-transcriptional level upon stress exposure and relief from stress. We have therefore set-up a RT-qPCR-based platform for high-throughput transcriptional profiling of a large set of chromatin genes. We find that the expression of a large fraction of these genes is regulated by cold. In addition, we reveal an induction of several DNA and histone demethylase genes and certain histone variants after plants have been shifted back to ambient temperature (deacclimation), suggesting a role in the memory of cold acclimation. We also re-analyse large scale transcriptomic datasets for transcriptional regulation and alternative splicing (AS) of chromatin genes, uncovering an unexpected level of regulation of these genes, particularly at the splicing level. This includes several vernalization regulating genes whose AS results in cold-regulated protein diversity. Overall, we provide a profiling platform for the analysis of chromatin regulatory genes and integrative analyses of their regulation, suggesting a dynamic regulation of key chromatin genes in response to low temperature stress.

plant biology↗

bioRxiv2019

Brunner syndrome associated MAOA dysfunction in human iPSC derived dopaminergic neurons results in dysregulated NMDAR expression and increased network activity

Monoamine oxidase A (MAOA) is an enzyme that catalyzes the degradation of dopamine, noradrenaline, and serotonin. Regulation of monoamine neurotransmitter abundance through MAOA activity strongly affects motor control, emotion, and cognitive function. Mutations in MAOA cause Brunner Syndrome, which is characterized by impulsive aggressive behavior and mild intellectual disability (ID). The impaired MAOA activity in Brunner Syndrome patients results in bioamine aberration, but it is currently unknown how this affects neuronal function. MAOA is highly expressed in serotonergic and dopaminergic neurons, and dysfunction of both neurotransmission systems is associated with aggressive behavior in mice and humans. Research has so far mainly focused on the serotonergic system. Here, we generated human induced pluripotent stem cell-derived induced dopaminergic neurons (iDANs) from individuals with known MAOA mutations, to investigate MAOA-dependent effects on dopamine neuronal function in the context of Brunner Syndrome. We assessed iDAN lines from three patients and combined data from morphological analysis, gene expression, single-cell electrophysiology, and network analysis using micro-electrode arrays (MEAs). We observed mutation-dependent functional effects as well as overlapping changes in iDAN morphology. The most striking effect was a clear increase in N-methyl-D-aspartate (NMDA) receptor mRNA expression in all patient lines. A marked increase was also seen in coordinated network activity (network bursts) on the MEA in all patient lines, while single-cell intrinsic properties and spontaneous excitatory postsynaptic currents activity appeared normal. Together, our data indicate that dysfunction of MAOA leads to increased coordinated network activity in iDANs, possibly caused by increased synaptic NMDA receptor expression.

neuroscience↗

bioRxiv2019

Measurement of skeletal muscle fiber contractility with high-speed traction microscopy

We describe a technique for simultaneous quantification of the contractile forces and cytosolic calcium dynamics of muscle fibers embedded in three-dimensional biopolymer gels. We derive a scaling law for linear elastic matrices such as basement membrane extract hydrogels (Matrigel) that allows us to measure contractile force from the shape of the relaxed and contracted muscle cell and the Youngs modulus of the matrix, without further knowledge of the matrix deformations surrounding the cell and without performing computationally intensive inverse force reconstruction algorithms. We apply our method to isolated mouse flexor digitorum brevis (FDB) fibers that are embedded in 10 mg/ml Matrigel. Upon electrical stimulation, individual FDB fibers show twitch forces of 0.37 {micro}N {+/-} 0.15 {micro}N and tetanic forces (100 Hz stimulation frequency) of 2.38 {micro}N {+/-} 0.71 {micro}N, corresponding to a tension of 0.44 kPa {+/-} 0.25 kPa and 2.53 kPa {+/-} 1.17 kPa, respectively. Contractile forces of FDB fibers increase in response to caffeine and the troponin-calcium-stabilizer Tirasemtiv, similar to responses measured in whole muscle. From simultaneous high-speed measurements of cell length changes and cytosolic calcium concentration using confocal line scanning at a frequency of 2048 Hz, we show that twitch and tetanic force responses to electric pulses follow the low-pass filtered calcium signal. In summary, we present a technically simple high speed and high throughput method for measuring contractile forces and cytosolic calcium dynamics of single muscle fibers. We expect that our method will help to reduce preparation time, costs, and the number of sacrificed animals needed for experiments such as drug testing.\n\nStatement of significanceWe describe a high speed, high throughput method for the simultaneous measurement of contractile force and cytoplasmic calcium dynamics following electrical pulse stimulation of muscle fibers embedded in a 3-dimensional biopolymer matrix. In contrast to the classical approach of attaching muscle fibers to a force-transducer, our method allows for a highly efficient, parallel analysis of large numbers of fibers under different treatment conditions.

bioengineering↗

bioRxiv2019

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

Epilepsy, 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.

neuroscience↗

bioRxiv2019

Neuronal network dysfunction in a human model for Kleefstra syndrome mediated by enhanced NMDAR signaling

Epigenetic regulation of gene transcription plays a critical role in neural network development and in the etiology of Intellectual Disability (ID) and Autism Spectrum Disorder (ASD). However, little is known about the mechanisms by which epigenetic dysregulation leads to neural network defects. Kleefstra syndrome (KS), caused by mutation in the histone methyltransferase EHMT1, is a neurodevelopmental disorder with the clinical features of both ID and ASD. To study the impact of decreased EHMT1 function in human cells, we generated excitatory cortical neurons from induced pluripotent stem (iPS) cells derived from KS patients. In addition, we created an isogenic set by genetically editing healthy iPS cells. Characterization of the neurons at the single-cell and neuronal network level revealed consistent discriminative properties that distinguished EHMT1-mutant from wildtype neurons. Mutant neuronal networks exhibited network bursting with a reduced rate, longer duration, and increased temporal irregularity compared to control networks. We show that these changes were mediated by the upregulation of the NMDA receptor (NMDAR) subunit 1 and correlate with reduced deposition of the repressive H3K9me2 mark, the catalytic product of EHMT1, at the GRIN1 promoter. Furthermore, we show that EHMT1 deficiency in mice leads to similar neuronal network impairments and increased NMDAR function. Finally, we could rescue the KS patient-derived neuronal network phenotypes by pharmacological inhibition of NMDARs. Together, our results demonstrate a direct link between EHMT1 deficiency in human neurons and NMDAR hyperfunction, providing the basis for a more targeted therapeutic approach to treating KS.

neuroscience↗

bioRxiv2018

Distinct pathogenic genes causing intellectual disability and autism exhibit overlapping effects on neuronal network development

An intriguing question in medical biology is how mutations in functionally distinct genes can lead to similar clinical phenotypes. For example, patients with mutations in distinct epigenetic regulators EHMT1, MBD5, MLL3 or SMARCB1 share the core clinical features of intellectual disability (ID), autism spectrum disorder (ASD) and facial dysmorphisms. To elucidate how these phenotypic similarities are reflected by convergence at the molecular, cellular and neuronal network level, we directly compared the effects of their loss of function in neurons. Interestingly, knockdown of each gene resulted in hyperactive neuronal networks with altered patterns of synchronized activity. At the single-cell level, we found genotype-specific changes in intrinsic excitability and excitatory-inhibitory balance, but in all cases leading to increased excitability. Congruent with our physiological findings, we identified dysregulated genes that converge on biological and cellular pathways related to neuronal excitability and synaptic function, including genes previously implicated in ID/ASD. Yet, our data suggests that the common cellular phenotypes depend on the ensemble of dysregulated genes engaged in neuronal excitability rather than the direction of transcriptional changes of individual genes. The demonstration of increasing convergence from molecular pathways to neuronal networks may be a paradigm for other types of ID/ASD.

neuroscience↗

bioRxiv2017

PWO1 interacts with PcG proteins and histones to regulate Arabidopsis flowering and development.

Polycomb-group (PcG) proteins mediate epigenetic gene regulation by setting H3K27me3 via Polycomb Repressive Complex 2 (PRC2). In plants, it is largely unclear how PcG proteins are recruited to their target genes.\n\nHere, we identified the PWWP-DOMAIN INTERACTOR OF POLYCOMBS1 (PWO1) protein which interacts with all three Arabidopsis PRC2 histone methyltransferases and is required for keeping full H3 occupancy at several Arabidopsis genes. PWO1 localizes and recruits CLF to nuclear speckles in tobacco nuclei, suggesting a role in spatial organization of PcG regulation. PWO1 belongs to a gene family with three members acting redundantly: pwo1 pwo2 pwo3 triple mutants are seedling lethal and show shoot and root meristem arrest, while pwo1 single mutants are early flowering. Interestingly, PWO1s PWWP domain confers binding to histones, which is reduced by a point mutation in a highly conserved residue of this domain and blocked by phosphorylation of H3S28. PWO1 carrying this mutation is not able to fully complement the pwo1 pwo2 pwo3 triple mutant, indicating the requirement of this domain for PWO1 in vivo activity. Thus, the PWO family may present a novel class of histone readers which are involved in recruiting PcG proteins to subnuclear domains and in promoting Arabidopsis development.

plant biology↗

bioRxiv2017

PWWP INTERACTOR OF POLYCOMBS (PWO1) links PcG-mediated gene repression to the nuclear lamina in Arabidopsis.

Polycomb group (PcG) proteins facilitate chromatin-mediated gene repression through the modification of histone tails in a wide range of eukaryotes, including plants and animals. One of the PcG protein complexes, Polycomb Repressive Complex 2 (PRC2), promotes repressive chromatin formation via tri-methylation of lysine-27 on histone H3 (H3K27me3). The animal PRC2 is implicated in impacting subnuclear distribution of chromatin as its complex components and H3K27me3 are functionally connected with the nuclear lamina (NL) - a peripheral protein mesh that resides underneath the inner nuclear membrane (INM) and consists of lamins and lamina-associated proteins. In contrast to animals, NL in plants has an atypical structure and its association with PRC2-mediated gene repression is largely unknown. Here, we present a connection between lamin-like protein, CROWDED NUCLEI 1 (CRWN1), and a novel PRC2-associated component, PWWP INTERACTOR OF POLYCOMBS 1 (PWO1), in Arabidopsis thaliana. We show that PWO1 and CRWN1 proteins associate physically with each other, act in the same pathway to maintain nuclear morphology and control expression of similar set of target genes. Moreover, we demonstrate that PWO1 proteins form speckle-like foci located partially at the subnuclear periphery in Nicotiana benthamiana and Arabidopsis thaliana. Ultimately, as CRWN1 and PWO1 are plant-specific, our results argue that plants developed an equivalent, rather than homologous, mechanism of linking PRC2-mediated chromatin repression and nuclear lamina.

molecular biology↗