Regulation of synaptic connectivity, including long-term depression (LTD), allows proper tuning of cellular signalling processes within brain circuitry. In the cerebellum, a key centre for motor coordination, a positive feedback loop that includes mitogen-activated protein kinases (MAPKs) is required for proper temporal control of LTD at cerebellar Purkinje cell synapses. Here we report that the tyrosine-specific MAPK-phosphatase PTPRR plays a role in coordinating the activity of this regulatory loop.

The ability to generate human induced pluripotent stem cells (iPSCs) from somatic cells provides tremendous promises for regenerative medicine and its use has widely increased over recent years. However, reprogramming efficiencies remain low and chromosomal instability and tumorigenic potential are concerns in the use of iPSCs, especially in clinical settings. Therefore, reprogramming methods have been under development to generate safer iPSCs with higher efficiency and better quality. Developments have mainly focused on the somatic cell source, the cocktail of reprogramming factors, the delivery method used to introduce reprogramming factors and culture conditions to maintain the generated iPSCs. This review discusses the developments on these topics and briefly discusses pros and cons of iPSCs in comparison with human embryonic stem cells generated from somatic cell nuclear transfer.

Heterozygous mutations or deletions in the human Euchromatin histone methyltransferase 1 (EHMT1) gene cause Kleefstra syndrome, a neurodevelopmental disorder that is characterized by autistic-like features and severe intellectual disability (ID). Neurodevelopmental disorders including ID and autism may be related to deficits in activity-dependent wiring of brain circuits during development. Although Kleefstra syndrome has been associated with dendritic and synaptic defects in mice and Drosophila, little is known about the role of EHMT1 in the development of cortical neuronal networks. Here we used micro-electrode arrays and whole-cell patch-clamp recordings to investigate the impact of EHMT1 deficiency at the network and single cell level. We show that EHMT1 deficiency impaired neural network activity during the transition from uncorrelated background action potential firing to synchronized network bursting. Spontaneous bursting and excitatory synaptic currents were transiently reduced, whereas miniature excitatory postsynaptic currents were not affected. Finally, we show that loss of function of EHMT1 ultimately resulted in less regular network bursting patterns later in development. These data suggest that the developmental impairments observed in EHMT1-deficient networks may result in a temporal misalignment between activity-dependent developmental processes thereby contributing to the pathophysiology of Kleefstra syndrome.

Koolen-de Vries syndrome (KdVS) is a multi-system disorder characterized by intellectual disability, friendly behavior, and congenital malformations. The syndrome is caused either by microdeletions in the 17q21.31 chromosomal region or by variants in the KANSL1 gene. The reciprocal 17q21.31 microduplication syndrome is associated with psychomotor delay, and reduced social interaction. To investigate the pathophysiology of 17q21.31 microdeletion and microduplication syndromes, we generated three mouse models: 1) the deletion (Del/+); or 2) the reciprocal duplication (Dup/+) of the 17q21.31 syntenic region; and 3) a heterozygous Kansl1 (Kans1+/-) model. We found altered weight, general activity, social behaviors, object recognition, and fear conditioning memory associated with craniofacial and brain structural changes observed in both Del/+ and Dup/+ animals. By investigating hippocampus function, we showed synaptic transmission defects in Del/+ and Dup/+ mice. Mutant mice with a heterozygous loss-of-function mutation in Kansl1 displayed similar behavioral and anatomical phenotypes compared to Del/+ mice with the exception of sociability phenotypes. Genes controlling chromatin organization, synaptic transmission and neurogenesis were upregulated in the hippocampus of Del/+ and Kansl1+/- animals. Our results demonstrate the implication of KANSL1 in the manifestation of KdVS phenotypes and extend substantially our knowledge about biological processes affected by these mutations. Clear differences in social behavior and gene expression profiles between Del/+ and Kansl1+/- mice suggested potential roles of other genes affected by the 17q21.31 deletion. Together, these novel mouse models provide new genetic tools valuable for the development of therapeutic approaches.

Intellectual disability (ID) disorders are genetically and phenotypically highly heterogeneous and present a major challenge in clinical genetics and medicine. Although many genes involved in ID have been identified, the etiology is unknown in most affected individuals. Moreover, the function of most genes associated with ID remains poorly characterized. Evidence is accumulating that the control of gene transcription through epigenetic modification of chromatin structure in neurons has an important role in cognitive processes and in the etiology of ID. However, our understanding of the key molecular players and mechanisms in this process is highly fragmentary. Here, we identify a chromatin-modification module that underlies a recognizable form of ID, the Kleefstra syndrome phenotypic spectrum (KSS). In a cohort of KSS individuals without mutations in EHMT1 (the only gene known to be disrupted in KSS until now), we identified de novo mutations in four genes, MBD5, MLL3, SMARCB1, and NR1I3, all of which encode epigenetic regulators. Using Drosophila, we demonstrate that MBD5, MLL3, and NR1I3 cooperate with EHMT1, whereas SMARCB1 is known to directly interact with MLL3. We propose a highly conserved epigenetic network that underlies cognition in health and disease. This network should allow the design of strategies to treat the growing group of ID pathologies that are caused by epigenetic defects.

Presenilins (PS) are proteins involved in the pathogenesis of autosomal-dominant familial cases of Alzheimer's disease. Mutations in PS are known to induce specific alterations in cellular Ca2+ signaling which might be involved in the pathogenesis of neurodegenerative diseases. Mouse embryonic fibroblasts (MEF) deficient in PS1 and PS2 (PS DKO) as well as the latter rescued with PS1 (Rescue), were used to investigate the underlying mechanism of these alterations in Ca2+ signaling. PS DKO cells were characterized by a decrease in the [Ca2+]ER as measured by ER-targeted aequorin luminescence and an increased level of type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). The lower [Ca2+]ER was associated with an increase in a Ca2+ leak from the ER. The increased IP3R1 expression and the concomitant changes in ER Ca2+ handling were reversed in the Rescue cells. Moreover using RNA-interference mediated reduction of IP3R1 we could demonstrate that the up-regulation of this isoform was responsible for the increased Ca2+ leak and the lowered [Ca2+]ER PS DKO cells. Finally, we show that the decreased [Ca2+]ER in PS DKO cells was protective against apoptosis.

Neural activity is essential for the maturation of sensory systems. In the rodent primary somatosensory cortex (S1), high extracellular serotonin (5-HT) levels during development impair neural transmission between the thalamus and cortical input layer IV (LIV). Rodent models of impaired 5-HT transporter (SERT) function show disruption in their topological organization of S1 and in the expression of activity-regulated genes essential for inhibitory cortical network formation. It remains unclear how such alterations affect the sensory information processing within cortical LIV. Using serotonin transporter knockout (Sert-/-) rats, we demonstrate that high extracellular serotonin levels are associated with impaired feedforward inhibition (FFI), fewer perisomatic inhibitory synapses, a depolarized GABA reversal potential and reduced expression of KCC2 transporters in juvenile animals. At the neural population level, reduced FFI increases the excitatory drive originating from LIV, facilitating evoked representations in the supragranular layers II/III. The behavioral consequence of these changes in network excitability is faster integration of the sensory information during whisker-based tactile navigation, as Sert-/- rats require fewer whisker contacts with tactile targets and perform object localization with faster reaction times. These results highlight the association of serotonergic homeostasis with formation and excitability of sensory cortical networks, and consequently with sensory perception.

Macroautophagy (hereafter referred to as 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 remodeling 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 SOD1 (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 GRIA/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 toward 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.Abbreviations: APO: apocynin; ATG: autophagy related; BAF: bafilomycin A1; BSO: buthionine sulfoximine; CV: coefficient of variation; DIV: days in vitro; H4K16ac: histone 4 lysine 16 acetylation; iPSC: induced-pluripotent stem cell; KANSL1: KAT8 regulatory NSL complex subunit 1; KdVS: Koolen-de Vries Syndrome; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEA: micro-electrode array; MTOR: mechanistic target of rapamycin kinase; NSL complex: nonspecific lethal complex; 8-oxo-dG: 8-hydroxydesoxyguanosine; RAP: rapamycin; ROS: reactive oxygen species; sEPSCs: spontaneous excitatory postsynaptic currents; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; SYN: synapsin; WRT: wortmannin.

We generated induced excitatory neurons (iNeurons, iNs) from chimpanzee, bonobo, and human stem cells by expressing the transcription factor neurogenin-2 (NGN2). Single-cell RNA sequencing showed that genes involved in dendrite and synapse development are expressed earlier during iNs maturation in the chimpanzee and bonobo than the human cells. In accordance, during the first 2 weeks of differentiation, chimpanzee and bonobo iNs showed repetitive action potentials and more spontaneous excitatory activity than human iNs, and extended neurites of higher total length. However, the axons of human iNs were slightly longer at 5 weeks of differentiation. The timing of the establishment of neuronal polarity did not differ between the species. Chimpanzee, bonobo, and human neurites eventually reached the same level of structural complexity. Thus, human iNs develop slower than chimpanzee and bonobo iNs, and this difference in timing likely depends on functions downstream of NGN2.

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

N-acetylneuraminic acid (sialic acid) is present in large quantities in the brain and plays a crucial role in brain development, learning, and memory formation. How sialic acid contributes to brain development is not fully understood. The purpose of this study was to determine the effects of reduced sialylation on network formation in human iPSC-derived neurons (iNeurons). Using targeted mass spectrometry and antibody binding, we observed an increase in free sialic acid and polysialic acid during neuronal development, which was disrupted by treatment of iNeurons with a synthetic inhibitor of sialic acid biosynthesis. Sialic acid inhibition disturbed synapse formation and network formation on microelectrode array (MEA), showing short but frequent (network) bursts and an overall lower firing rate, and higher percentage of random spikes. This study shows that sialic acid is necessary for neuronal network formation during human neuronal development and provides a physiologically relevant model to study the role of sialic acid in patient-derived iNeurons.

We implicated the X-chromosome THOC2 gene, which encodes the largest subunit of the highly-conserved TREX (Transcription-Export) complex, in a clinically complex neurodevelopmental disorder with intellectual disability as the core phenotype. To study the molecular pathology of this essential eukaryotic gene, we generated a mouse model based on a hypomorphic Thoc2 exon 37-38 deletion variant of a patient with ID, speech delay, hypotonia, and microcephaly. The Thoc2 exon 37-38 deletion male (Thoc2Δ/Y) mice recapitulate the core phenotypes of THOC2 syndrome including smaller size and weight, and significant deficits in spatial learning, working memory and sensorimotor functions. The Thoc2Δ/Y mouse brain development is significantly impacted by compromised THOC2/TREX function resulting in R-loop accumulation, DNA damage and consequent cell death. Overall, we suggest that perturbed R-loop homeostasis, in stem cells and/or differentiated cells in mice and the patient, and DNA damage-associated functional alterations are at the root of THOC2 syndrome.

Remodeling of chromatin accessibility is necessary for successful reprogramming of fibroblasts to neurons. However, it is still not fully known which transcription factors can induce a neuronal chromatin accessibility profile when overexpressed in fibroblasts. To identify such transcription factors, we used ATAC-sequencing to generate differential chromatin accessibility profiles between human fibroblasts and iNeurons, an in vitro neuronal model system obtained by overexpression of Neurog2 in induced pluripotent stem cells (iPSCs). We found that the ONECUT transcription factor sequence motif was strongly associated with differential chromatin accessibility between iNeurons and fibroblasts. All three ONECUT transcription factors associated with this motif (ONECUT1, ONECUT2 and ONECUT3) induced a neuron-like morphology and expression of neuronal genes within two days of overexpression in fibroblasts. We observed widespread remodeling of chromatin accessibility; in particular, we found that chromatin regions that contain the ONECUT motif were in- or lowly accessible in fibroblasts and became accessible after the overexpression of ONECUT1, ONECUT2 or ONECUT3. There was substantial overlap with iNeurons, still, many regions that gained accessibility following ONECUT overexpression were not accessible in iNeurons. Our study highlights both the potential and challenges of ONECUT-based direct neuronal reprogramming.

At glutamatergic synapses, local endocytic recycling of AMPA receptors (AMPARs) is important for the supply of a mobile pool of AMPARs required for synaptic potentiation. This local recycling of AMPARs critically relies on the presence of an endocytic zone (EZ) near the postsynaptic density (PSD). The precise mechanisms that couple the EZ to the PSD still remain largely elusive, with the large GTPase Dynamin-3 and the multimeric PSD adaptor protein Homer1 as the two main players identified. Here, we demonstrate that a physical interaction between the X-linked mental retardation protein oligophrenin-1 (OPHN1) and Homer1b/c is crucial for the positioning of the EZ adjacent to the PSD, and present evidence that this interaction is important for OPHN1's role in controlling activity-dependent strengthening of excitatory synapses in the rat hippocampus. Disruption of the OPHN1-Homer1b/c interaction causes a displacement of EZs from the PSD, along with impaired AMPAR recycling and reduced AMPAR accumulation at synapses, in both basal conditions and conditions that can induce synaptic potentiation. Together, our findings unveil a novel role for OPHN1 as an interaction partner of Homer1b/c in spine EZ positioning, and provide new mechanistic insight into how genetic deficits in OPHN1 can lead to impaired synapse maturation and plasticity.

Kleefstra syndrome, a disease with intellectual disability, autism spectrum disorders and other developmental defects is caused in humans by haploinsufficiency of EHMT1. Although EHMT1 and its paralog EHMT2 were shown to be histone methyltransferases responsible for deposition of the di-methylated H3K9 (H3K9me2), the exact nature of epigenetic dysfunctions in Kleefstra syndrome remains unknown. Here, we found that the epigenome of Ehmt1+/- adult mouse brain displays a marked increase of H3K9me2/3 which correlates with impaired expression of protocadherins, master regulators of neuronal diversity. Increased H3K9me3 was present already at birth, indicating that aberrant methylation patterns are established during embryogenesis. Interestingly, we found that Ehmt2+/- mice do not present neither the marked increase of H3K9me2/3 nor the cognitive deficits found in Ehmt1+/- mice, indicating an evolutionary diversification of functions. Our finding of increased H3K9me3 in Ehmt1+/- mice is the first one supporting the notion that EHMT1 can quench the deposition of tri-methylation by other Histone methyltransferases, ultimately leading to impaired neurocognitive functioning. Our insights into the epigenetic pathophysiology of Kleefstra syndrome may offer guidance for future developments of therapeutic strategies for this disease.

Kleefstra syndrome (KS) is a neurodevelopmental disorder caused by mutations in the histone methyltransferase EHMT1. 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. Neuronal networks of patient-derived cells exhibit network bursting with a reduced rate, longer duration, and increased temporal irregularity compared to control networks. We show that these changes are mediated by upregulation of NMDA receptor (NMDAR) subunit 1 correlating with reduced deposition of the repressive H3K9me2 mark, the catalytic product of EHMT1, at the GRIN1 promoter. In mice EHMT1 deficiency leads to similar neuronal network impairments with increased NMDAR function. Finally, we rescue the KS patient-derived neuronal network phenotypes by pharmacological inhibition of NMDARs. Summarized, we demonstrate a direct link between EHMT1 deficiency and NMDAR hyperfunction in human neurons, providing a potential basis for more targeted therapeutic approaches for KS.

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.

Protocadherin-19 (PCDH19) is a synaptic cell-adhesion molecule encoded by X-linked PCDH19, a gene linked with epilepsy. Here, we report a synapse-to-nucleus signaling pathway through which PCDH19 bridges neuronal activity with gene expression. In particular, we describe the NMDA receptor (NMDAR)-dependent proteolytic cleavage of PCDH19, which leads to the generation of a PCDH19 C-terminal fragment (CTF) able to enter the nucleus. We demonstrate that PCDH19 CTF associates with chromatin and with the chromatin remodeler lysine-specific demethylase 1 (LSD1) and regulates expression of immediate-early genes (IEGs). Our results are consistent with a model whereby PCDH19 favors maintenance of neuronal homeostasis via negative feedback regulation of IEG expression and provide a key to interpreting PCDH19-related hyperexcitability.

Activity in the healthy brain relies on a 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 patient, has typically been hampered by methodological limitations. Cadherin-13 (CDH13) has been associated with autism and attention-deficit/hyperactivity disorder. CDH13 localizes 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 MEF2C (PV-precursor marker protein) 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-β1 and Integrin-β3, 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.

Parkinson's disease is a common incurable neurodegenerative disease. The identification of genetic variants via genome-wide association studies has considerably advanced our understanding of the Parkinson's disease genetic risk. Understanding the functional significance of the risk loci is now a critical step towards translating these genetic advances into an enhanced biological understanding of the disease. Impaired mitophagy is a key causative pathway in familial Parkinson's disease, but its relevance to idiopathic Parkinson's disease is unclear. We used a mitophagy screening assay to evaluate the functional significance of risk genes identified through genome-wide association studies. We identified two new regulators of PINK1-dependent mitophagy initiation, KAT8 and KANSL1, previously shown to modulate lysine acetylation. These findings suggest PINK1-mitophagy is a contributing factor to idiopathic Parkinson's disease. KANSL1 is located on chromosome 17q21 where the risk associated gene has long been considered to be MAPT. While our data do not exclude a possible association between the MAPT gene and Parkinson's disease, they provide strong evidence that KANSL1 plays a crucial role in the disease. Finally, these results enrich our understanding of physiological events regulating mitophagy and establish a novel pathway for drug targeting in neurodegeneration.