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| Examples of terminal dendrite dynamics. Both wild-type and mutant neurons were observed extending, retracting, branching, and pruning. (A-D) Representative images of dendrite behavior. Top panels, low magnification view of individual neurons, visualized by imaging of tdTomato cytoplasmic fills. Areas within the red boxes are magnified in the time-lapse panels below each neuron. Dashed lines indicate the positions of each dendrite tip at the beginning of imaging. White arrows indicate the position of the dendrite tip in each frame. Growth cones were considered the tips of dendrites, rather than individual filopodia. Imaging times are displayed relative to the start of imaging (0:00) and span 4 h. Intensities are colored as in Figure 1. Scale bars, 10 µm. (A) wildtype dendrite extending and remaining terminal (white arrows). (B) Wildtype dendrite retracting and remaining terminal (white arrows). (C) GluN2B 724t dendrite emerging, growing, and then retracting (white arrows). (D) GluN2B 724t dendrite retracting and pruning (white arrows).
Source publication
Mutations in GRIN2B, which encodes the GluN2B subunit of NMDA receptors, lead to autism spectrum disorders (ASD), but the pathophysiological mechanisms remain unclear. Recently, we showed that a GluN2B variant that is associated with severe ASD (GluN2B724t) impairs dendrite morphogenesis. To determine which aspects of dendrite growth are affected b...
Context in source publication
Context 1
... with our previous observations in more mature neurons (Sceniak et al., 2019), 5-9 DIV neurons expressing GluN2B 724t appeared to have fewer, as well as shorter, dendrites than GluN2B WT neurons ( Figure 1B). Time-lapse images were collected over 4 h ( Figure 1B; Supplementary Videos 1, 2), and terminal dendrites were tracked for the following fates: extension, retraction, pruning, and branching (Figure 2). Terminal dendrites were analyzed because they are the segments that are actively growing. ...
Citations
... It also reduces its trafficking to dendrites and synapses. The mutation also interferes with spine density and morphology, resulting in fewer and smaller spines [53,54]. ...
NMDA receptors (NMDARs) are a subtype of ionotropic glutamate receptors that mediate excitatory neurotransmission and synaptic plasticity in the brain. NMDARs play important roles in various normal brain functions such as learning, memory, and cognition, but also contribute to the pathogenesis of several developmental, neurological, and psychiatric disorders. Alterations in NMDARs can result in either hypo- or hyperfunction of NMDARs, which can impair neuronal viability, synaptic efficacy, and network oscillations. In this review, we summarize the current knowledge on the involvement of NMDA receptors in Alzheimer’s disease, autism spectrum disorder, epilepsy, and schizophrenia. We also highlight the potential therapeutic strategies that target NMDAR modulation and dysfunction in these disorders.
... GluN2B containing receptors have a lower open probability, slower deactivation kinetics and an increased sensitivity to glutamate and glycine compared to GluN2A containing receptors. 12,13 In the context of neurodevelopmental disorders, rodent in vitro data demonstrates that pathogenic GRIN2B variants alter NMDAR function 14 , neuronal migration 15 , dendrite morphogenesis 16,17 and synaptic density 16,18 , which likely contribute to the aberrant circuit properties that manifest in GRIN2B-related neurodevelopmental disorders. ...
Pathogenic mutations in GRIN2B are an important cause of severe neurodevelopmental disorders resulting in epilepsy, autism and intellectual disability. GRIN2B encodes the GluN2B subunit of N-methyl-D-aspartate receptors (NMDARs), which are ionotropic glutamate receptors critical for normal development of the nervous system and synaptic plasticity. Here, we characterized a novel GRIN2B heterozygous knockout rat model with 24-hour EEG recordings. We found rats heterozygous for the deletion ( Grin2b +/- ) had a higher incidence of spontaneous absence seizures than wild-type rats ( Grin2b +/+ ). Spike and wave discharges, the electrographic correlate of absences seizures, were longer in duration and displayed increased higher overall spectral power in Grin2b +/- animals than those in Grin2b +/+ . Heterozygous mutant rats also had abnormal sleep-wake brain state dynamics over the circadian cycle. Specifically, we identified a reduction in total rapid eye movement sleep and, altered distributions of non-rapid eye movement sleep and wake epochs, when compared to controls. This was accompanied by an increase in overall spectral power during non-rapid eye movement sleep in Grin2b +/- . The sleep-wake phenotypes were largely uncorrelated to the incidence of spike and wave discharges. We then tested the antiseizure efficacy of ethosuximide, a T-type voltage-gated calcium channel blocker used in the treatment of absence seizures, and memantine, a noncompetitive NMDAR antagonist currently explored as a mono or adjunctive treatment option in NMDAR related neurodevelopmental disorders. Ethosuximide reduced the number and duration of spike and wave discharges, while memantine did not affect the number of spike and wave discharges but reduced their duration. These results highlight two potential therapeutic options for GRIN2B related epilepsy. Our data shows the new rat GRIN2B haploinsufficiency model exhibits clinically relevant phenotypes. As such, it could prove crucial in deciphering underlying pathological mechanisms and developing new therapeutically translatable strategies for GRIN2B neurodevelopmental disorders.
... Decreased expression of GluN2B, a subunit of NMDA receptors, has been associated with shortening of neuronal dendrites (Bahry et al., 2021) and reduced synaptic connection, which impairs the development of normal and functional circuits. In the study by Favre et al. (2015), after applying the EE protocol, an increase in GluN2B expression and reversal of the social deficit were found, evidencing the benefit of EE and associating this marker with the pathogenesis of ASD (Roullet et al., 2010;Etherton et al., 2011;Bostrom et al., 2015;De Filippis et al., 2015). ...
Environmental enrichment (EE) is known to produce experience-dependent changes in the brains and behaviors of rodents, and it has therefore been widely used to study neurodevelopmental disorders, including autism. Current studies show significant protocol variation, such as the presence of running wheels, number of cagemates, duration of enrichment, and the age of the animals at the beginning and end of the enrichment interventions. EE has been shown to have prominent positive effects in animal models of idiopathic and syndromic autism, but little is known about the ideal type of EE and the most efficient protocols for reversing autism spectrum disorder (ASD) behaviors modeled in rodents. This review presents evidence that social enrichment is the most effective way to rescue typical behaviors, and that variables such as onset, duration, and type of induction in the ASD model are important for EE success. Understanding which EE protocols are most beneficial for reversing ASD behaviors modeled in rodents opens up possibilities for the potential treatment of neuropsychiatric disorders characterized by behavioral deficits, such as autism.
... Glutamate receptor ionotropic NMDA2B (Grin2b) is linked to epileptic encephalopathy, ASD, and other neurological disorders in which Grin2b haploinsufficiency is often an important factor [45,87]. GluN2B deletion disrupts protein-dependent homeostatic plasticity, according to in vitro electrophysiological investigations. ...
Autism spectrum disorder (ASD) is a heterogeneous, behaviorally defined neurodevelopmental disorder. Over the past two decades, the prevalence of autism spectrum disorders has progressively increased, however, no clear diagnostic markers and specifically targeted medications for autism have emerged. As a result, neurobehavioral abnormalities, neurobiological alterations in ASD, and the development of novel ASD pharmacological therapy necessitate multidisciplinary collaboration. In this review, we discuss the development of multiple animal models of ASD to contribute to the disease mechanisms of ASD, as well as new studies from multiple disciplines to assess the behavioral pathology of ASD. In addition, we summarize and highlight the mechanistic advances regarding gene transcription, RNA and non-coding RNA translation, abnormal synaptic signaling pathways, epigenetic post-translational modifications, brain-gut axis, immune inflammation and neural loop abnormalities in autism to provide a theoretical basis for the next step of precision therapy. Furthermore, we review existing autism therapy tactics and limits and present challenges and opportunities for translating multidisciplinary knowledge of ASD into clinical practice.
... This gene is involved in a variety of neurodevelopmental diseases such as West syndrome and intellectual impairment with focal epilepsy [3] and may be linked to infantile spasms and Lennox-Gastaut syndrome [4]. GRIN2B variations have been identified in GRIN2B-related neurodevelopmental disorders, such as ID [5], ASD [6,7], Atypical Rett Syndrome & intractable epilepsy [8], encephalopathy [9], developmental delay or macrocephaly [10], schizophrenia [11], obsessive compulsive disorder [12], and cerebral visual impairment [13], causing cells to create an inactive GluN2B protein or prevent them from making any GluN2B protein. Researchers are uncertain how improper NMDA receptor activation compromises typical brain growth and development or why excessive or inadequate activity manifests in neurological complications in people with GRIN2B-related neurodevelopmental disorders. ...
The GluN2B subunit of N-methyl-D-aspartate receptors plays an important role in the physiology of different neurodevelopmental diseases. Genetic variations in the GluN2B coding gene (GRIN2B) have consistently been linked to West syndrome, intellectual impairment with focal epilepsy, developmental delay, macrocephaly, corticogenesis, brain plasticity, as well as infantile spasms and Lennox-Gastaut syndrome. It is unknown, however, how GRIN2B genetic variation impacts protein function. We determined the cumulative pathogenic impact of GRIN2B variations on healthy participants using a computational approach. We looked at all of the known mutations and calculated the impact of single nucleotide polymorphisms on GRIN2B, which encodes the GluN2B protein. The pathogenic effect, functional impact, conservation analysis, post-translation alterations, their driving residues, and dynamic behaviors of deleterious nsSNPs on protein models were then examined. Four polymorphisms were identified as phylogenetically conserved PTM drivers and were related to structural and functional impact: rs869312669 (p.Thr685Pro), rs387906636 (p.Arg682Cys), rs672601377 (p.Asn615Ile), and rs1131691702 (p.Ser526Pro). The combined impact of protein function is accounted for by the calculated stability , compactness, and total globularity score. GluN2B hydrogen occupancy was positively associated with protein stability, and solvent-accessible surface area was positively related to globulari-ty. Furthermore, there was a link between GluN2B protein folding, movement, and function, indicating that both putative high and low local movements were linked to protein function. Multiple GRIN2B genetic variations are linked to gene expression, phylogenetic conservation, PTMs, and protein instability behavior in neurodevelopmental diseases. These findings suggest the relevance of GRIN2B genetic variations in neurodevelopmental problems.
... An alternative explanation may center around a possible WDFY3 requirement for cytoskeletal organization, as widespread axonal defects in homozygous mutants suggest (unpublished observations and [64]). Interestingly, decreased dendritic complexity in Wdfy3 mutant neurons agrees with findings in other ASD risk genes [65][66][67][68][69], has been observed postmortem in the hippocampi of autism cases [70], and may be a point of convergence between ASD and Rett syndrome [44]. ...
Background
Proper cerebral cortical development depends on the tightly orchestrated migration of newly born neurons from the inner ventricular and subventricular zones to the outer cortical plate. Any disturbance in this process during prenatal stages may lead to neuronal migration disorders (NMDs), which can vary in extent from focal to global. Furthermore, NMDs show a substantial comorbidity with other neurodevelopmental disorders, notably autism spectrum disorders (ASDs). Our previous work demonstrated focal neuronal migration defects in mice carrying loss-of-function alleles of the recognized autism risk gene WDFY3 . However, the cellular origins of these defects in Wdfy3 mutant mice remain elusive and uncovering it will provide critical insight into WDFY3 -dependent disease pathology.
Methods
Here, in an effort to untangle the origins of NMDs in Wdfy3 lacZ mice, we employed mosaic analysis with double markers (MADM). MADM technology enabled us to genetically distinctly track and phenotypically analyze mutant and wild-type cells concomitantly in vivo using immunofluorescent techniques.
Results
We revealed a cell autonomous requirement of WDFY3 for accurate laminar positioning of cortical projection neurons and elimination of mispositioned cells during early postnatal life. In addition, we identified significant deviations in dendritic arborization, as well as synaptic density and morphology between wild type, heterozygous, and homozygous Wdfy3 mutant neurons in Wdfy3 -MADM reporter mice at postnatal stages.
Limitations
While Wdfy3 mutant mice have provided valuable insight into prenatal aspects of ASD pathology that remain inaccessible to investigation in humans, like most animal models, they do not a perfectly replicate all aspects of human ASD biology. The lack of human data makes it indeterminate whether morphological deviations described here apply to ASD patients or some of the other neurodevelopmental conditions associated with WDFY3 mutation.
Conclusions
Our genetic approach revealed several cell autonomous requirements of WDFY3 in neuronal development that could underlie the pathogenic mechanisms of WDFY3 -related neurodevelopmental conditions. The results are also consistent with findings in other ASD animal models and patients and suggest an important role for WDFY3 in regulating neuronal function and interconnectivity in postnatal life.
... This suggests that the impact of GluN2B mutations depends on the developmental stage. At a cellular level, heterozygosity for mutations in GRIN2B have also been shown to affect neuronal differentiation [75], and overexpression of GluN2B mutants has been shown to affect dendritic morphogenesis [76,77], both effects which we likely evade in our postnatal (< P7), single-cell genetic manipulation. Nonetheless, our data suggest that very different deficits in NMDA-EPSCs may be apparent depending on developmental stage. ...
GRIN2B mutations are rare but often associated with patients having severe neurodevelopmental disorders with varying range of symptoms such as intellectual disability, developmental delay and epilepsy. Patient symptoms likely arise from mutations disturbing the role that the encoded NMDA receptor subunit, GluN2B, plays at neuronal connections in the developing nervous system. In this study, we investigated the cell-autonomous effects of putative gain- (GoF) and loss-of-function (LoF) missense GRIN2B mutations on excitatory synapses onto CA1 pyramidal neurons in organotypic hippocampal slices. In the absence of both native GluN2A and GluN2B subunits, functional incorporation into synaptic NMDA receptors was attenuated for GoF mutants, or almost eliminated for LoF GluN2B mutants. NMDA-receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) from synaptic GoF GluN1/2B receptors had prolonged decays consistent with their functional classification. Nonetheless, in the presence of native GluN2A, molecular replacement of native GluN2B with GoF and LoF GluN2B mutants all led to similar functional incorporation into synaptic receptors, more rapidly decaying NMDA-EPSCs and greater inhibition by TCN-201, a selective antagonist for GluN2A-containing NMDA receptors. Mechanistic insight was gained from experiments in HEK293T cells, which revealed that GluN2B GoF mutants slowed deactivation in diheteromeric GluN1/2B, but not triheteromeric GluN1/2A/2B receptors. We also show that a disease-associated missense mutation, which severely affects surface expression, causes opposing effects on NMDA-EPSC decay and charge transfer when introduced into GluN2A or GluN2B. Finally, we show that having a single null Grin2b allele has only a modest effect on NMDA-EPSC decay kinetics. Our results demonstrate that functional incorporation of GoF and LoF GluN2B mutants into synaptic receptors and the effects on EPSC decay times are highly dependent on the presence of triheteromeric GluN1/2A/2B NMDA receptors, thereby influencing the functional classification of NMDA receptor variants as GoF or LoF mutations. These findings highlight the complexity of interpreting effects of disease-causing NMDA receptor missense mutations in the context of neuronal function.
... The identification of GRIN variants in pediatric patients is significant, as individuals GRIN mutations and epilepsy refractory to standard anti-convulsants have been successfully treated with the FDA-approved NMDAR antagonists memantine, ketamine, and dextromethorphan (Pierson et al., 2014;Li et al., 2016;Amador et al., 2020;Xu et al., 2021). Although mutations in four genes encoding NMDAR subunits (GRIN1, GRIN2A, GRIN2B, and GRIN2D) have been linked to human disease (Carvill et al., 2013;Li et al., 2016;Liu et al., 2017;Li et al., 2019;Bahry et al., 2021;Xu et al., 2021), mutations in GRIN2A account for the majority of disease-linked variants (46%) (Myers et al., 2019;Strehlow et al., 2019). Pathogenic variants cluster in the highly conserved agonist binding domains as well as transmembrane and linker domains, which are highly intolerant to genetic variation (Swanger et al., 2016;Ogden et al., 2017;Strehlow et al., 2019). ...
Mutations in N-methyl-d-aspartate receptors (NMDAR) subunits have been implicated in a growing number of human neurodevelopmental disorders. Previously, a de novo mutation in GRIN2A, encoding the GluN2A subunit, was identified in a patient with severe epilepsy and developmental delay. This missense mutation, which leads to GluN2A-P552R, produces significant dendrotoxicity in transfected rodent cortical neurons, as evidenced by pronounced dendritic blebbing. This injurious process can be prevented by treatment with the NMDA antagonist memantine. Given the increasing use of FDA approved NMDA antagonists to treat patients with GRIN mutations, who may have seizures refractory to traditional anti-epileptic drugs, we investigated whether additional NMDA antagonists were effective in attenuating neurotoxicity associated with GluN2A-P552R expression. Intriguingly, we found that while treatment with memantine can effectively block GluN2A-P552R-mediated dendrotoxicity, treatment with ketamine does not, despite the fact that both drugs work as open NMDAR channel blockers. Interestingly, we found that neurons expressing GluN2A-P552R were more vulnerable to an excitotoxic insult—an effect that, in this case, could be equally rescued by both memantine and ketamine. These findings suggest that GluN2A-P552R induced dendrotoxicity and increased vulnerability to excitotoxic stress are mediated through two distinct mechanisms. The differences between memantine and ketamine in halting GluN2A-P552R dendrotoxicity could not be explained by NMDA antagonist induced changes in MAP or Src kinase activation, previously shown to participate in NMDA-induced excitotoxicity. Our findings strongly suggest that not all NMDA antagonists may be of equal clinical utility in treating GRIN2A-mediated neurological disorders, despite a shared mechanism of action.
Proper growth and branching of dendrites are crucial for adequate central nervous system (CNS) functioning. The neuronal dendritic geometry determines the mode and quality of information processing. Any defects in dendrite development will disrupt neuronal circuit formation, affecting brain function. Besides cell-intrinsic programmes, extrinsic factors regulate various aspects of dendritic development. Among these extrinsic factors are extracellular molecular signals which can shape the dendrite architecture during early development. This review will focus on extrinsic factors regulating dendritic growth during early neuronal development, including neurotransmitters, neurotrophins, extracellular matrix proteins, contact-mediated ligands, and secreted and diffusible cues. How these extracellular molecular signals contribute to dendritic growth has been investigated in developing nervous systems using different species, different areas within the CNS, and different neuronal types. The response of the dendritic tree to these extracellular molecular signals can result in growth-promoting or growth-limiting effects, and it depends on the receptor subtype, receptor quantity, receptor efficiency, the animal model used, the developmental time windows, and finally, the targeted signal cascade. This article reviews our current understanding of the role of various extracellular signals in the establishment of the architecture of the dendrites.
