Socrates: A Novel N-Ethyl-N-nitrosourea-Induced Mouse Mutant with Audiogenic Epilepsy.

Epilepsy is one of the common neurological diseases that affects not only adults but also infants and children. Because epilepsy has been studied for a long time, there are several pharmacologically effective anticonvulsants, which, however, are not suitable as therapy for all patients. The genesis of epilepsy has been extensively investigated in terms of its occurrence after injury and as a concomitant disease with various brain diseases, such as tumors, ischemic events, etc. However, in the last decades, there are multiple reports that both genetic and epigenetic factors play an important role in epileptogenesis. Therefore, there is a need for further identification of genes and loci that can be associated with higher susceptibility to epileptic seizures. Use of mouse knockout models of epileptogenesis is very informative, but it has its limitations. One of them is due to the fact that complete deletion of a gene is not, in many cases, similar to human epilepsy-associated syndromes. Another approach to generating mouse models of epilepsy is N-Ethyl-N-nitrosourea (ENU)-directed mutagenesis. Recently, using this approach, we generated a novel mouse strain, soc (socrates, formerly s8-3), with epileptiform activity. Using molecular biology methods, calcium neuroimaging, and immunocytochemistry, we were able to characterize the strain. Neurons isolated from soc mutant brains retain the ability to differentiate in vitro and form a network. However, soc mutant neurons are characterized by increased spontaneous excitation activity. They also demonstrate a high degree of Ca2+ activity compared to WT neurons. Additionally, they show increased expression of NMDA receptors, decreased expression of the Ca2+-conducting GluA2 subunit of AMPA receptors, suppressed expression of phosphoinositol 3-kinase, and BK channels of the cytoplasmic membrane involved in protection against epileptogenesis. During embryonic and postnatal development, the expression of several genes encoding ion channels is downregulated in vivo, as well. Our data indicate that soc mutation causes a disruption of the excitation-inhibition balance in the brain, and it can serve as a mouse model of epilepsy.

Srsf1 and Elavl1 act antagonistically on neuronal fate choice in the developing neocortex by controlling TrkC receptor isoform expression.

The seat of higher-order cognitive abilities in mammals, the neocortex, is a complex structure, organized in several layers. The different subtypes of principal neurons are distributed in precise ratios and at specific positions in these layers and are generated by the same neural progenitor cells (NPCs), steered by a spatially and temporally specified combination of molecular cues that are incompletely understood. Recently, we discovered that an alternatively spliced isoform of the TrkC receptor lacking the kinase domain, TrkC-T1, is a determinant of the corticofugal projection neuron (CFuPN) fate. Here, we show that the finely tuned balance between TrkC-T1 and the better known, kinase domain-containing isoform, TrkC-TK+, is cell type-specific in the developing cortex and established through the antagonistic actions of two RNA-binding proteins, Srsf1 and Elavl1. Moreover, our data show that Srsf1 promotes the CFuPN fate and Elavl1 promotes the callosal projection neuron (CPN) fate in vivo via regulating the distinct ratios of TrkC-T1 to TrkC-TK+. Taken together, we connect spatio-temporal expression of Srsf1 and Elavl1 in the developing neocortex with the regulation of TrkC alternative splicing and transcript stability and neuronal fate choice, thus adding to the mechanistic and functional understanding of alternative splicing in vivo.

Reactivation of corticogenesis-related transcriptional factors BCL11B and SATB2 after ischemic lesion of the adult mouse brain.

The aim of this study was to characterize expression of corticogenesis-related transcription factors BCL11B and SATB2 after brain ischemic lesion in the adult mice, and to analyze their correlation to the subsequent brain recovery. Ischemic brain lesion was induced by transient middle cerebral artery occlusion followed by reperfusion, and the animals with ischemic lesion were compared to the sham controls. Progression of the brain damage and subsequent recovery was longitudinally monitored structurally, by magnetic resonance imaging, and functionally, by neurological deficit assessment. Seven days after the ischemic injury the brains were isolated and analyzed by immunohistochemistry. The results showed higher expression in the brain of both, BCL11B and SATB2 in the animals with ischemic lesion compared to the sham controls. The co-expression of both markers, BCL11B and SATB2, increased in the ischemic brains, as well as the co-expression of BCL11B with the beneficial transcriptional factor ATF3 but not its co-expression with detrimental HDAC2. BCL11B was mainly implicated in the ipsilateral and SATB2 in the contralateral brain hemisphere, and their level in these regions correlated with the functional recovery rate. The results indicate that the reactivation of corticogenesis-related transcription factors BCL11B and SATB2 is beneficial after brain ischemic lesion.

The murine ortholog of Kaufman oculocerebrofacial syndrome gene Ube3b is crucial for the maintenance of the excitatory synapses in the young adult stage.

Kaufman oculocerebrofacial syndrome (KOS) is an autosomal recessive developmental disorder. Inactivating mutations in UBE3B, an E3 ubiquitin ligase gene are causative for KOS. We have reported that towards postnatal week three, its murine ortholog, Ube3b, acts as a negative regulator of the number of dendritic spines. In this study, we investigated the role of Ube3b at the synapse in the young adult mice. With an improved estimation method, images from the hippocampal CA1 and CA2 regions acquired with 3D Stimulated Emission Depletion (3D-STED) microscopy were used to quantify the excitatory synapse numbers. In the young adult mice, the excitatory synapse density was decreased in brain-specific Ube3b conditional knockout mice as compared to the control. Our results indicate the novel role of Ube3b in the maintenance of synapse numbers in the young adult period.

TrkB-dependent EphrinA reverse signaling regulates callosal axon fasciculate growth downstream of Neurod2/6.

Abnormal development of corpus callosum is relatively common and causes a broad spectrum of cognitive impairments in humans. We use acallosal Neurod2/6-deficient mice to study callosal axon guidance within the ipsilateral cerebral cortex. Initial callosal tracts form but fail to traverse the ipsilateral cingulum and are not attracted towards the midline in the absence of Neurod2/6. We show that the restoration of Ephrin-A4 (EfnA4) expression in the embryonic neocortex of Neurod2/6-deficient embryos is sufficient to partially rescue targeted callosal axon growth towards the midline. EfnA4 cannot directly mediate reverse signaling within outgrowing axons, but it forms co-receptor complexes with TrkB (Ntrk2). The ability of EfnA4 to rescue the guided growth of a subset of callosal axons in Neurod2/6-deficient mice is abolished by the co-expression of dominant negative TrkBK571N (kinase-dead) or TrkBY515F (SHC-binding deficient) variants, but not by TrkBY816F (PLCγ1-binding deficient). Additionally, EphA4 is repulsive to EfnA4-positive medially projecting axons in organotypic brain slice culture. Collectively, we suggest that EfnA4-mediated reverse signaling acts via TrkB-SHC and is required for ipsilateral callosal axon growth accuracy towards the midline downstream of Neurod family factors.

Molecular mechanisms of corpus callosum development: a four-step journey.

The Corpus Callosum (CC) is a bundle of axons connecting the cerebral hemispheres. It is the most recent structure to have appeared during evolution of placental mammals. Its development is controlled by a very complex interplay of many molecules. In humans it contains almost 80% of all commissural axons in the brain. The formation of the CC can be divided into four main stages, each controlled by numerous intracellular and extracellular molecular factors. First, a newborn neuron has to specify an axon, leave proliferative compartments, the Ventricular Zone (VZ) and Subventricular Zone (SVZ), migrate through the Intermediate Zone (IZ), and then settle at the Cortical Plate (CP). During the second stage, callosal axons navigate toward the midline within a compact bundle. Next stage is the midline crossing into contralateral hemisphere. The last step is targeting a defined area and synapse formation. This review provides an insight into these four phases of callosal axons development, as well as a description of the main molecular players involved.

A critical period of translational control during brain development at codon resolution.

Translation modulates the timing and amplification of gene expression after transcription. Brain development requires uniquely complex gene expression patterns, but large-scale measurements of translation directly in the prenatal brain are lacking. We measure the reactants, synthesis and products of mRNA translation spanning mouse neocortex neurogenesis, and discover a transient window of dynamic regulation at mid-gestation. Timed translation upregulation of chromatin-binding proteins like Satb2, which is essential for neuronal subtype differentiation, restricts protein expression in neuronal lineages despite broad transcriptional priming in progenitors. In contrast, translation downregulation of ribosomal proteins sharply decreases ribosome biogenesis, coinciding with a major shift in protein synthesis dynamics at mid-gestation. Changing activity of eIF4EBP1, a direct inhibitor of ribosome biogenesis, is concurrent with ribosome downregulation and affects neurogenesis of the Satb2 lineage. Thus, the molecular logic of brain development includes the refinement of transcriptional programs by translation. Modeling of the developmental neocortex translatome is provided as an open-source searchable resource at https://shiny.mdc-berlin.de/cortexomics .

Interleukin-10 restores glutamate receptor-mediated Ca2+-signaling in brain circuits under loss of Sip1 transcription factor.

OBJECTIVE: This study aimed to investigate the connection between the mutation of the Sip1 transcription factor and impaired Ca2+-signaling, which reflects changes in neurotransmission in the cerebral cortex in vitro. METHODS: We used mixed neuroglial cortical cell cultures derived from Sip1 mutant mice. The cells were loaded with a fluorescent ratiometric calcium-sensitive probe Fura-2 AM and epileptiform activity was modeled by excluding magnesium ions from the external media or adding a GABA(A) receptor antagonist, bicuculline. Intracellular calcium dynamics were recorded using fluorescence microscopy. To identify the level of gene expression, the Real-Time PCR method was used. RESULTS: It was found that cortical neurons isolated from homozygous (Sip1fl/fl) mice with the Sip1 mutation demonstrate suppressed Ca2+ signals in models of epileptiform activity in vitro. Wild-type cortical neurons are characterized by synchronous high-frequency and high-amplitude Ca2+ oscillations occurring in all neurons of the network in response to Mg2+-free medium and bicuculline. But cortical Sip1fl/fl neurons only single Ca2+ pulses or attenuated Ca2+ oscillations are recorded and only in single neurons, while most of the cell network does not respond to these stimuli. This signal deficiency of Sip1fl/fl neurons correlates with a suppressed expression level of the genes encoding the subunits of NMDA, AMPA, and KA receptors; protein kinases PKA, JNK, CaMKII; and also the transcription factor Hif1α. These negative effects were partially abolished when Sip1fl/fl neurons are grown in media with anti-inflammatory cytokine IL-10. IL-10 increases the expression of the above-mentioned genes but not to the level of expression in wild-type. At the same time, the amplitudes of Ca2+ signals increase in response to the selective agonists of NMDA, AMPA and KA receptors, and the proportion of neurons responding with Ca2+ oscillations to a Mg2+-free medium and bicuculline increases. CONCLUSION: IL-10 restores neurotransmission in neuronal networks with the Sip1 mutation by regulating the expression of genes encoding signaling proteins.

PCSK9 deficiency alters brain lipid composition without affecting brain development and function.

PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor (LDLR) in the liver, hereby preventing removal of LDL cholesterol from the circulation. Accordingly, PCSK9 inhibitory antibodies and siRNA potently reduce LDL cholesterol to unprecedented low levels and are approved for treatment of hypercholesterolemia. In addition, PCSK9 inactivation alters the levels of several other circulating lipid classes and species. Brain function is critically influenced by cholesterol and lipid composition. However, it remains unclear how the brain is affected long-term by the reduction in circulating lipids as achieved with potent lipid lowering therapeutics such as PCSK9 inhibitors. Furthermore, it is unknown if locally expressed PCSK9 affects neuronal circuits through regulation of receptor levels. We have studied the effect of lifelong low peripheral cholesterol levels on brain lipid composition and behavior in adult PCSK9 KO mice. In addition, we studied the effect of PCSK9 on neurons in culture and in vivo in the developing cerebral cortex. We found that PCSK9 reduced LDLR and neurite complexity in cultured neurons, but neither PCSK9 KO nor overexpression affected cortical development in vivo. Interestingly, PCSK9 deficiency resulted in changes of several lipid classes in the adult cortex and cerebellum. Despite the observed changes, PCSK9 KO mice had unchanged behavior compared to WT controls. In conclusion, our findings demonstrate that altered PCSK9 levels do not compromise brain development or function in mice, and are in line with clinical trials showing that PCSK9 inhibitors have no adverse effects on cognitive function.

Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B.

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.

TrkC-T1, the Non-Catalytic Isoform of TrkC, Governs Neocortical Progenitor Fate Specification by Inhibition of MAP Kinase Signaling.

Neocortical projection neurons are generated by neural progenitor cells (NPCs) within the ventricular and subventricular zone. While early NPCs can give rise to both deep and upper layer neurons, late progenitors are restricted to upper layer neurogenesis. The molecular mechanisms controlling the differentiation potential of early versus late NPCs are unknown. Here, we report a novel function for TrkC-T1, the non-catalytic isoform of the neurotrophin receptor TrkC, that is distinct from TrkC-TK+, the full-length isoform. We provide direct evidence that TrkC-T1 regulates the switch in NPC fate from deep to upper layer neuron production. Elevated levels of TrkC-T1 in early NPCs promote the generation of deep layer neurons. Conversely, downregulation of TrkC-T1 in these cells promotes upper layer neuron fate. Furthermore, we show that TrkC-T1 exerts this control by interaction with the signaling adaptor protein ShcA. TrkC-T1 prevents the phosphorylation of Shc and the downstream activation of the MAP kinase (Erk1/2) pathway. In vivo manipulation of the activity of ShcA or Erk1/2, directly affects cortical neuron cell fate. We thus show that the generation of upper layer neurons by late progenitors is dependent on the downregulation of TrkC-T1 in late progenitor cells and the resulting activation of the ShcA/Erk1/2 pathway.

Adhesion dynamics in the neocortex determine the start of migration and the post-migratory orientation of neurons.

The neocortex is stereotypically organized into layers of excitatory neurons arranged in a precise parallel orientation. Here we show that dynamic adhesion both preceding and following radial migration is essential for this organization. Neuronal adhesion is regulated by the Mowat-Wilson syndrome-associated transcription factor Zeb2 (Sip1/Zfhx1b) through direct repression of independent adhesion pathways controlled by Neuropilin-1 (Nrp1) and Cadherin-6 (Cdh6). We reveal that to initiate radial migration, neurons must first suppress adhesion to the extracellular matrix. Zeb2 regulates the multipolar stage by transcriptional repression of Nrp1 and thereby downstream inhibition of integrin signaling. Upon completion of migration, neurons undergo an orientation process that is independent of migration. The parallel organization of neurons within the neocortex is controlled by Cdh6 through atypical regulation of integrin signaling via its RGD motif. Our data shed light on the mechanisms that regulate initiation of radial migration and the postmigratory orientation of neurons during neocortical development.

The Role of Neurod Genes in Brain Development, Function, and Disease.

Transcriptional regulation is essential for the correct functioning of cells during development and in postnatal life. The basic Helix-loop-Helix (bHLH) superfamily of transcription factors is well conserved throughout evolution and plays critical roles in tissue development and tissue maintenance. A subgroup of this family, called neural lineage bHLH factors, is critical in the development and function of the central nervous system. In this review, we will focus on the function of one subgroup of neural lineage bHLH factors, the Neurod family. The Neurod family has four members: Neurod1, Neurod2, Neurod4, and Neurod6. Available evidence shows that these four factors are key during the development of the cerebral cortex but also in other regions of the central nervous system, such as the cerebellum, the brainstem, and the spinal cord. We will also discuss recent reports that link the dysfunction of these transcription factors to neurological disorders in humans.

Expanding the phenotype of NUP85 mutations beyond nephrotic syndrome to primary autosomal recessive microcephaly and Seckel syndrome spectrum disorders.

Primary autosomal recessive microcephaly and Seckel syndrome spectrum disorders (MCPH-SCKS) include a heterogeneous group of autosomal recessive inherited diseases characterized by primary (congenital) microcephaly, the absence of visceral abnormalities, and a variable degree of cognitive impairment, short stature and facial dysmorphism. Recently, biallelic variants in the nuclear pore complex (NPC) component nucleoporin 85 gene (NUP85) were reported to cause steroid-resistant nephrotic syndrome (SRNS). Here, we report biallelic variants in NUP85 in two pedigrees with an MCPH-SCKS phenotype spectrum without SRNS, thereby expanding the phenotypic spectrum of NUP85-linked diseases. Structural analysis predicts the identified NUP85 variants cause conformational changes that could have an effect on NPC architecture or on its interaction with other NUPs. We show that mutant NUP85 is, however, associated with a reduced number of NPCs but unaltered nucleocytoplasmic compartmentalization, abnormal mitotic spindle morphology, and decreased cell viability and proliferation in one patient's cells. Our results also indicate the link of common cellular mechanisms involved in MCPH-SCKS spectrum disorders and NUP85-associated diseases. In addition to the previous studies, our results broaden the phenotypic spectrum of NUP85-linked human disease and propose a role for NUP85 in nervous system development.

Ablation of Vti1a/1b Triggers Neural Progenitor Pool Depletion and Cortical Layer 5 Malformation in Late-embryonic Mouse Cortex.

Cortical morphogenesis entails several neurobiological events, including proliferation and differentiation of progenitors, migration of neuroblasts, and neuronal maturation leading to functional neural circuitry. These neurodevelopmental processes are delicately regulated by many factors. Endosomal SNAREs have emerged as formidable modulators of neuronal growth, aside their well-known function in membrane/vesicular trafficking. However, our understanding of their influence on cortex formation is limited. Here, we report that the SNAREs Vti1a and Vti1b (Vti1a/1b) are critical for proper cortical development. Following null mutation of Vti1a/1b in mouse, the late-embryonic mutant cortex appeared dysgenic, and the cortical progenitors therein were depleted beyond normal. Notably, cortical layer 5 (L5) is distinctively disorganized in the absence of Vti1a/1b. The latter defect, coupled with an overt apoptosis of Ctip2-expressing L5 neurons, likely contributed to the substantial loss of corticospinal and callosal projections in the Vti1a/1b-deficient mouse brain. These findings suggest that Vti1a/1b serve key neurodevelopmental functions during cortical histogenesis, which when mechanistically elucidated, can lend clarity to how endosomal SNAREs regulate brain development, or how their dysfunction may have implications for neurological disorders.

Olig3 regulates early cerebellar development.

The mature cerebellum controls motor skill precision and participates in other sophisticated brain functions that include learning, cognition, and speech. Different types of GABAergic and glutamatergic cerebellar neurons originate in temporal order from two progenitor niches, the ventricular zone and rhombic lip, which express the transcription factors Ptf1a and Atoh1, respectively. However, the molecular machinery required to specify the distinct neuronal types emanating from these progenitor zones is still unclear. Here, we uncover the transcription factor Olig3 as a major determinant in generating the earliest neuronal derivatives emanating from both progenitor zones in mice. In the rhombic lip, Olig3 regulates progenitor cell proliferation. In the ventricular zone, Olig3 safeguards Purkinje cell specification by curtailing the expression of Pax2, a transcription factor that suppresses the Purkinje cell differentiation program. Our work thus defines Olig3 as a key factor in early cerebellar development.

The murine ortholog of Kaufman oculocerebrofacial syndrome protein Ube3b regulates synapse number by ubiquitinating Ppp3cc.

Kaufman oculocerebrofacial syndrome (KOS) is a severe autosomal recessive disorder characterized by intellectual disability, developmental delays, microcephaly, and characteristic dysmorphisms. Biallelic mutations of UBE3B, encoding for a ubiquitin ligase E3B are causative for KOS. In this report, we characterize neuronal functions of its murine ortholog Ube3b and show that Ube3b regulates dendritic branching in a cell-autonomous manner. Moreover, Ube3b knockout (KO) neurons exhibit increased density and aberrant morphology of dendritic spines, altered synaptic physiology, and changes in hippocampal circuit activity. Dorsal forebrain-specific Ube3b KO animals show impaired spatial learning, altered social interactions, and repetitive behaviors. We further demonstrate that Ube3b ubiquitinates the catalytic γ-subunit of calcineurin, Ppp3cc, the overexpression of which phenocopies Ube3b loss with regard to dendritic spine density. This work provides insights into the molecular pathologies underlying intellectual disability-like phenotypes in a genetically engineered mouse model.

SNAP to attention: A SNARE complex regulates neuronal progenitor polarity.

SNARE vesicle targeting complex controls the polarity of neuronal progenitors. Kunii et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.201910080) show that the SNAP23-VAMP8-Syntaxin1B complex is required for membrane targeting of N-cadherin and formation of adherence junction complexes in radial glia neuronal progenitors, the major prerequisite of cell polarity establishment.

Protein Synthesis in the Developing Neocortex at Near-Atomic Resolution Reveals Ebp1-Mediated Neuronal Proteostasis at the 60S Tunnel Exit.

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.

Molecular Evolution, Neurodevelopmental Roles and Clinical Significance of HECT-Type UBE3 E3 Ubiquitin Ligases.

Protein ubiquitination belongs to the best characterized pathways of protein degradation in the cell; however, our current knowledge on its physiological consequences is just the tip of an iceberg. The divergence of enzymatic executors of ubiquitination led to some 600-700 E3 ubiquitin ligases embedded in the human genome. Notably, mutations in around 13% of these genes are causative of severe neurological diseases. Despite this, molecular and cellular context of ubiquitination remains poorly characterized, especially in the developing brain. In this review article, we summarize recent findings on brain-expressed HECT-type E3 UBE3 ligases and their murine orthologues, comprising Angelman syndrome UBE3A, Kaufman oculocerebrofacial syndrome UBE3B and autism spectrum disorder-associated UBE3C. We summarize evolutionary emergence of three UBE3 genes, the biochemistry of UBE3 enzymes, their biology and clinical relevance in brain disorders. Particularly, we highlight that uninterrupted action of UBE3 ligases is a sine qua non for cortical circuit assembly and higher cognitive functions of the neocortex.