Foxp2 loss of function increases striatal direct pathway inhibition via increased GABA release

JRvR is supported by an RUMC Junior Round grant from the Donders institute Ph.D. program, awarded to NNK and SCV. SCV is supported by a Marie Curie Career Integration Grant (PCIG12-GA-2012-333978) and by a Max Planck Research Group Award. SEF is supported by the Max Planck Society.Heterozygous mutations of the Forkhead-box protein 2 (FOXP2) gene in humans cause childhood apraxia of speech. Loss of Foxp2 in mice is known to affect striatal development and impair motor skills. However, it is unknown if striatal excitatory/inhibitory balance is affected during development and if the imbalance persists into adulthood. We investigated the effect of reduced Foxp2 expression, via a loss-of-function mutation, on striatal medium spiny neurons (MSNs). Our data show that heterozygous loss of Foxp2 decreases excitatory (AMPA receptor-mediated) and increases inhibitory (GABA receptor-mediated) currents in D1 dopamine receptor positive MSNs of juvenile and adult mice. Furthermore, reduced Foxp2 expression increases GAD67 expression, leading to both increased presynaptic content and release of GABA. Finally, pharmacological blockade of inhibitory activity in vivo partially rescues motor skill learning deficits in heterozygous Foxp2 mice. Our results suggest a novel role for Foxp2 in the regulation of striatal direct pathway activity through managing inhibitory drive.Publisher PDFPeer reviewe

Epigenetic Etiology of Intellectual Disability.

Intellectual disability (ID) is a prevailing neurodevelopmental condition associated with impaired cognitive and adaptive behaviors. Many chromatin-modifying enzymes and other epigenetic regulators have been genetically associated with ID disorders (IDDs). Here we review how alterations in the function of histone modifiers, chromatin remodelers, and methyl-DNA binding proteins contribute to neurodevelopmental defects and altered brain plasticity. We also discuss how progress in human genetics has led to the generation of mouse models that unveil the molecular etiology of ID, and outline the direction in which this field is moving to identify therapeutic strategies for IDDs. Importantly, because the chromatin regulators linked to IDDs often target common downstream genes and cellular processes, the impact of research in individual syndromes goes well beyond each syndrome and can also contribute to the understanding and therapy of other IDDs. Furthermore, the investigation of these disorders helps us to understand the role of chromatin regulators in brain development, plasticity, and gene expression, thereby answering fundamental questions in neurobiology

Basal ryanodine receptor activity suppresses autophagic flux

The inositol 1,4,5-trisphosphate receptors (IP3Rs) and intracellular Ca2+ signaling are critically involved in regulating different steps of autophagy, a lysosomal degradation pathway. The ryanodine receptors (RyR), intracellular Ca2+-release channels mainly expressed in excitable cell types including muscle and neurons, have however not yet been extensively studied in relation to autophagy. Yet, aberrant expression and excessive activity of RyRs in these tissues has been implicated in the onset of several diseases including Alzheimer’s disease, where impaired autophagy regulation contributes to the pathology. In this study, we determined whether pharmacological RyR inhibition could modulate autophagic flux in ectopic RyR-expressing models, like HEK293 cells and in cell types that endogenously express RyRs, like C2C12 myoblasts and primary hippocampal neurons. Importantly, RyR3 overexpression in HEK293 cells impaired the autophagic flux. Conversely, in all cell models tested, pharmacological inhibition of endogenous or ectopically expressed RyRs, using dantrolene or ryanodine, augmented autophagic flux by increasing lysosomal turn-over (number of autophagosomes and autolysosomes measured as mCherry-LC3 punctae/cell increased from 70.37 ± 7.81 in control HEK RyR3 cells to 111.18 ± 7.72 and 98.14 ± 7.31 after dantrolene and ryanodine treatments, respectively). Moreover, in differentiated C2C12 cells, transmission electron microscopy demonstrated that dantrolene treatment decreased the number of early autophagic vacuoles from 5.9 ± 2.97 to 1.8 ± 1.03 per cellular cross section. The modulation of the autophagic flux could be linked to the functional inhibition of RyR channels as both RyR inhibitors efficiently diminished the number of cells showing spontaneous RyR3 activity in the HEK293 cell model (from 41.14% ± 2.12 in control cells to 18.70% ± 2.25 and 9.74% ± 2.67 after dantrolene and ryanodine treatments, respectively). In conclusion, basal RyR-mediated Ca2+-release events suppress autophagic flux at the level of the lysosomes

Ryanodine receptors are targeted by anti-apoptotic Bcl-X-L involving its BH4 domain and Lys87 from its BH3 domain

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members target several intracellular Ca2+-transport systems. Bcl-2, via its N-terminal Bcl-2 homology (BH) 4 domain, inhibits both inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs), while Bcl-X-L, likely independently of its BH4 domain, sensitizes IP3Rs. It remains elusive whether Bcl-XL can also target and modulate RyRs. Here, Bcl-X-L co-immunoprecipitated with RyR3 expressed in HEK293 cells. Mammalian protein-protein interaction trap (MAPPIT) and surface plasmon resonance (SPR) showed that Bcl-XL bound to the central domain of RyR3 via its BH4 domain, although to a lesser extent compared to the BH4 domain of Bcl-2. Consistent with the ability of the BH4 domain of Bcl-X-L to bind to RyRs, loading the BH4-Bcl-X-L peptide into RyR3-overexpressing HEK293 cells or in rat hippocampal neurons suppressed RyR-mediated Ca2+ release. In silico superposition of the 3D-structures of Bcl-2 and Bcl-XL indicated that Lys87 of the BH3 domain of Bcl-XL could be important for interacting with RyRs. In contrast to Bcl-X-L, the Bcl-X-L(K87D) mutant displayed lower binding affinity for RyR3 and a reduced inhibition of RyR-mediated Ca2+ release. These data suggest that Bcl-X-L binds to RyR channels via its BH4 domain, but also its BH3 domain, more specific Lys87, contributes to the interaction

Unexpected Heterodivalent Recruitment of NOS1AP to nNOS Reveals Multiple Sites for Pharmacological Intervention in Neuronal Disease Models

The protein NOS1AP/CAPON mediates signaling from a protein complex of NMDA receptor, PSD95 and nNOS. The only stroke trial for neuroprotectants that showed benefit to patients targeted this ternary complex. NOS1AP/nNOS interaction regulates small GTPases, iron transport, p38MAPK-linked excitotoxicity, and anxiety. Moreover, the nos1ap gene is linked to disorders from schizophrenia, post-traumatic stress disorder, and autism to cardiovascular disorders and breast cancer. Understanding protein interactions required for NOS1AP function, therefore, has broad implications for numerous diseases. Here we show that the interaction of NOS1AP with nNOS differs radically from the classical PDZ docking assumed to be responsible. The NOS1AP PDZ motif does not bind nNOS as measured by multiple methods. In contrast, full-length NOS1AP forms an unusually stable interaction with nNOS. We mapped the discrepancy between full-length and C-terminal PDZ motif to a novel internal region we call the ExF motif. The C-terminal PDZ motif, although neither sufficient nor necessary for binding, nevertheless promotes the stability of the complex. It therefore potentially affects signal transduction and suggests that functional interaction of nNOS with NOS1AP might be targetable at two distinct sites. We demonstrate that excitotoxic pathways can be regulated, in cortical neuron and organotypic hippocampal slice cultures from rat, either by the previously described PDZ ligand TAT-GESV or by the ExF motif-bearing region of NOS1AP, even when lacking the critical PDZ residues as long as the ExF motif is intact and not mutated. This previously unrecognized heterodivalent interaction of nNOS with NOS1AP may therefore provide distinct opportunities for pharmacological intervention in NOS1AP-dependent signaling and excitotoxicity.</p

A human in vitro neuronal model for studying homeostatic plasticity at the network level

Mechanisms that underlie homeostatic plasticity have been extensively investigated at single-cell levels in animal models, but are less well understood at the network level. Here, we used microelectrode arrays to characterize neuronal networks following induction of homeostatic plasticity in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with rat astrocytes. Chronic suppression of neuronal activity through tetrodotoxin (TTX) elicited a time-dependent network re-arrangement. Increased expression of AMPA receptors and the elongation of axon initial segments were associated with increased network excitability following TTX treatment. Transcriptomic profiling of TTX-treated neurons revealed up-regulated genes related to extracellular matrix organization, while down-regulated genes related to cell communication; also astrocytic gene expression was found altered. Overall, our study shows that hiPSC-derived neuronal networks provide a reliable in vitro platform to measure and characterize homeostatic plasticity at network and single-cell levels; this platform can be extended to investigate altered homeostatic plasticity in brain disorders.The work was supported by funding from the European Community’s Horizon 2020 Programme (H2020/2014–2020) under grant agreement no. 728018 (Eat2beNICE) (to B.F.); ERA-NET NEURON-102 SYNSCHIZ grant (NWO) 013-17-003 4538 (to D.S.); China Scholarship Council 201906100038 (to X.Y.); ISCIII /MINECO (PT17/0009/0019) and FEDER (to A.E.C.); and M.M. was supported by an internal grant from the Donders Centre for Medical Neurosciences of the Radboud University Medical Center

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

Heterozygous loss-of-function (LoF) mutations in SETD1A, which encodes a subunit of histone H3 lysine 4 methyltransferase, cause a neurodevelopmental syndrome and increase the risk for schizophrenia. Using CRISPR-Cas9, we generate excitatory/inhibitory neuronal networks from human induced pluripotent stem cells with a SETD1A heterozygous LoF mutation (SETD1A+/−). Our data show that SETD1A haploinsufficiency results in morphologically increased dendritic complexity and functionally increased bursting activity. This network phenotype is primarily driven by SETD1A haploinsufficiency in glutamatergic neurons. In accordance with the functional changes, transcriptomic profiling reveals perturbations in gene sets associated with glutamatergic synaptic function. At the molecular level, we identify specific changes in the cyclic AMP (cAMP)/Protein Kinase A pathway pointing toward a hyperactive cAMP pathway in SETD1A+/− neurons. Finally, by pharmacologically targeting the cAMP pathway, we are able to rescue the network deficits in SETD1A+/− cultures. Our results demonstrate a link between SETD1A and the cAMP-dependent pathway in human neurons.publishedVersio

SCN1A-deficient excitatory neuronal networks display mutation-specific phenotypes

Dravet syndrome is a severe epileptic encephalopathy, characterized by (febrile) seizures, behavioural problems and developmental delay. Eighty per cent of patients with Dravet syndrome have a mutation in SCN1A, encoding Nav1.1. Milder clinical phenotypes, such as GEFS+ (generalized epilepsy with febrile seizures plus), can also arise from SCN1A mutations. Predicting the clinical phenotypic outcome based on the type of mutation remains challenging, even when the same mutation is inherited within one family. This clinical and genetic heterogeneity adds to the difficulties of predicting disease progression and tailoring the prescription of anti-seizure medication. Understanding the neuropathology of different SCN1A mutations may help to predict the expected clinical phenotypes and inform the selection of best-fit treatments. Initially, the loss of Na+-current in inhibitory neurons was recognized specifically to result in disinhibition and consequently seizure generation. However, the extent to which excitatory neurons contribute to the pathophysiology is currently debated and might depend on the patient clinical phenotype or the specific SCN1A mutation. To examine the genotype-phenotype correlations of SCN1A mutations in relation to excitatory neurons, we investigated a panel of patient-derived excitatory neuronal networks differentiated on multi-electrode arrays. We included patients with different clinical phenotypes, harbouring various SCN1A mutations, along with a family in which the same mutation led to febrile seizures, GEFS+ or Dravet syndrome. We hitherto describe a previously unidentified functional excitatory neuronal network phenotype in the context of epilepsy, which corresponds to seizurogenic network prediction patterns elicited by proconvulsive compounds. We found that excitatory neuronal networks were affected differently, depending on the type of SCN1A mutation, but did not segregate according to clinical severity. Specifically, loss-of-function mutations could be distinguished from missense mutations, and mutations in the pore domain could be distinguished from mutations in the voltage sensing domain. Furthermore, all patients showed aggravated neuronal network responses at febrile temperatures compared with controls. Finally, retrospective drug screening revealed that anti-seizure medication affected GEFS+ patient- but not Dravet patient-derived neuronal networks in a patient-specific and clinically relevant manner. In conclusion, our results indicate a mutation-specific excitatory neuronal network phenotype, which recapitulates the foremost clinically relevant features, providing future opportunities for precision therapies.</p

Increased GABAB receptor signaling in a rat model for schizophrenia

Contains fulltext : 167879.pdf (publisher's version ) (Open Access)Schizophrenia is a complex disorder that affects cognitive function and has been linked, both in patients and animal models, to dysfunction of the GABAergic system. However, the pathophysiological consequences of this dysfunction are not well understood. Here, we examined the GABAergic system in an animal model displaying schizophrenia-relevant features, the apomorphine-susceptible (APO-SUS) rat and its phenotypic counterpart, the apomorphine-unsusceptible (APO-UNSUS) rat at postnatal day 20-22. We found changes in the expression of the GABA-synthesizing enzyme GAD67 specifically in the prelimbic- but not the infralimbic region of the medial prefrontal cortex (mPFC), indicative of reduced inhibitory function in this region in APO-SUS rats. While we did not observe changes in basal synaptic transmission onto LII/III pyramidal cells in the mPFC of APO-SUS compared to APO-UNSUS rats, we report reduced paired-pulse ratios at longer inter-stimulus intervals. The GABAB receptor antagonist CGP 55845 abolished this reduction, indicating that the decreased paired-pulse ratio was caused by increased GABAB signaling. Consistently, we find an increased expression of the GABAB1 receptor subunit in APO-SUS rats. Our data provide physiological evidence for increased presynaptic GABAB signaling in the mPFC of APO-SUS rats, further supporting an important role for the GABAergic system in the pathophysiology of schizophrenia