Cell-type-resolved mosaicism reveals clonal dynamics of the human forebrain.

Debate remains around the anatomical origins of specific brain cell subtypes and lineage relationships within the human forebrain1-7. Thus, direct observation in the mature human brain is critical for a complete understanding of its structural organization and cellular origins. Here we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific mosaic variant barcode analysis. From four hemispheres and two different human neurotypical donors, we identified 287 and 780 mosaic variants, respectively, that were used to deconvolve clonal dynamics. Clonal spread and allele fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted than resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome transcriptome analysis at both a cell-type-specific and a single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of mosaic variants across 17 locations within one parietal lobe reveals that restriction of clonal spread in the anterior-posterior axis precedes restriction in the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus, cell-type-resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

Homozygous Mutations in CSF1R Cause a Pediatric-Onset Leukoencephalopathy and Can Result in Congenital Absence of Microglia.

Microglia are CNS-resident macrophages that scavenge debris and regulate immune responses. Proliferation and development of macrophages, including microglia, requires Colony Stimulating Factor 1 Receptor (CSF1R), a gene previously associated with a dominant adult-onset neurological condition (adult-onset leukoencephalopathy with axonal spheroids and pigmented glia). Here, we report two unrelated individuals with homozygous CSF1R mutations whose presentation was distinct from ALSP. Post-mortem examination of an individual with a homozygous splice mutation (c.1754-1G>C) demonstrated several structural brain anomalies, including agenesis of corpus callosum. Immunostaining demonstrated almost complete absence of microglia within this brain, suggesting that it developed in the absence of microglia. The second individual had a homozygous missense mutation (c.1929C>A [p.His643Gln]) and presented with developmental delay and epilepsy in childhood. We analyzed a zebrafish model (csf1rDM) lacking Csf1r function and found that their brains also lacked microglia and had reduced levels of CUX1, a neuronal transcription factor. CUX1+ neurons were also reduced in sections of homozygous CSF1R mutant human brain, identifying an evolutionarily conserved role for CSF1R signaling in production or maintenance of CUX1+ neurons. Since a large fraction of CUX1+ neurons project callosal axons, we speculate that microglia deficiency may contribute to agenesis of the corpus callosum via reduction in CUX1+ neurons. Our results suggest that CSF1R is required for human brain development and establish the csf1rDM fish as a model for microgliopathies. In addition, our results exemplify an under-recognized form of phenotypic expansion, in which genes associated with well-recognized, dominant conditions produce different phenotypes when biallelically mutated.

ATP1A2- and ATP1A3-associated early profound epileptic encephalopathy and polymicrogyria.

Constitutional heterozygous mutations of ATP1A2 and ATP1A3, encoding for two distinct isoforms of the Na+/K+-ATPase (NKA) alpha-subunit, have been associated with familial hemiplegic migraine (ATP1A2), alternating hemiplegia of childhood (ATP1A2/A3), rapid-onset dystonia-parkinsonism, cerebellar ataxia-areflexia-progressive optic atrophy, and relapsing encephalopathy with cerebellar ataxia (all ATP1A3). A few reports have described single individuals with heterozygous mutations of ATP1A2/A3 associated with severe childhood epilepsies. Early lethal hydrops fetalis, arthrogryposis, microcephaly, and polymicrogyria have been associated with homozygous truncating mutations in ATP1A2. We investigated the genetic causes of developmental and epileptic encephalopathies variably associated with malformations of cortical development in a large cohort and identified 22 patients with de novo or inherited heterozygous ATP1A2/A3 mutations. We characterized clinical, neuroimaging and neuropathological findings, performed in silico and in vitro assays of the mutations' effects on the NKA-pump function, and studied genotype-phenotype correlations. Twenty-two patients harboured 19 distinct heterozygous mutations of ATP1A2 (six patients, five mutations) and ATP1A3 (16 patients, 14 mutations, including a mosaic individual). Polymicrogyria occurred in 10 (45%) patients, showing a mainly bilateral perisylvian pattern. Most patients manifested early, often neonatal, onset seizures with a multifocal or migrating pattern. A distinctive, 'profound' phenotype, featuring polymicrogyria or progressive brain atrophy and epilepsy, resulted in early lethality in seven patients (32%). In silico evaluation predicted all mutations to be detrimental. We tested 14 mutations in transfected COS-1 cells and demonstrated impaired NKA-pump activity, consistent with severe loss of function. Genotype-phenotype analysis suggested a link between the most severe phenotypes and lack of COS-1 cell survival, and also revealed a wide continuum of severity distributed across mutations that variably impair NKA-pump activity. We performed neuropathological analysis of the whole brain in two individuals with polymicrogyria respectively related to a heterozygous ATP1A3 mutation and a homozygous ATP1A2 mutation and found close similarities with findings suggesting a mainly neural pathogenesis, compounded by vascular and leptomeningeal abnormalities. Combining our report with other studies, we estimate that ∼5% of mutations in ATP1A2 and 12% in ATP1A3 can be associated with the severe and novel phenotypes that we describe here. Notably, a few of these mutations were associated with more than one phenotype. These findings assign novel, 'profound' and early lethal phenotypes of developmental and epileptic encephalopathies and polymicrogyria to the phenotypic spectrum associated with heterozygous ATP1A2/A3 mutations and indicate that severely impaired NKA pump function can disrupt brain morphogenesis.

AUTS2 Regulates RNA Metabolism and Dentate Gyrus Development in Mice.

Human AUTS2 mutations are linked to a syndrome of intellectual disability, autistic features, epilepsy, and other neurological and somatic disorders. Although it is known that this unique gene is highly expressed in developing cerebral cortex, the molecular and developmental functions of AUTS2 protein remain unclear. Using proteomics methods to identify AUTS2 binding partners in neonatal mouse cerebral cortex, we found that AUTS2 associates with multiple proteins that regulate RNA transcription, splicing, localization, and stability. Furthermore, AUTS2-containing protein complexes isolated from cortical tissue bound specific RNA transcripts in RNA immunoprecipitation and sequencing assays. Deletion of all major functional isoforms of AUTS2 (full-length and C-terminal) by conditional excision of exon 15 caused breathing abnormalities and neonatal lethality when Auts2 was inactivated throughout the developing brain. Mice with limited inactivation of Auts2 in cerebral cortex survived but displayed abnormalities of cerebral cortex structure and function, including dentate gyrus hypoplasia with agenesis of hilar mossy neurons, and abnormal spiking activity on EEG. Also, RNA transcripts that normally associate with AUTS2 were dysregulated in mutant mice. Together, these findings indicate that AUTS2 regulates RNA metabolism and is essential for development of cerebral cortex, as well as subcortical breathing centers.

What Are the Double Lines of the Fetal Cavum Septi Pellucidi on Ultrasound?

OBJECTIVE: To demonstrate the significance of the double line appearance of the septi pellucidi laminae (SPL) on fetal ultrasound. METHOD: A total of 522 uncomplicated singleton pregnancies (15 to 39 weeks' gestational age) with fetal ultrasounds were enrolled. The presence of a single versus double line SP as well as measurement of the cavum septi pellucidi (CSP) was determined retrospectively. Ultrasound settings from the CSP images were recorded. Thickness of the SPL was measured in 20 ultrasound and 14 MRI cases; histology was reviewed from one neonate. Maternal BMI and gestational age were also recorded. RESULTS: The presence of double line SPL is a normal sonographic finding, seen in 47% (188/403) of normal fetuses. Thickness of the SPL in 10 cases with double line averaged 1.4 mm and in 10 cases with single line averaged 0.8 mm; MRI measurements were within 0.1 mm of the corresponding ultrasound measurements. Double line cavum was more often seen with mid-dynamic contrast range settings (5, 6) rather than high range settings (7-10) (P value <.05). The double line was only visualized on ultrasound when the angle of insonation was at or near perpendicular to the laminae; it was never visualized on coronal ultrasound imaging or MRI imaging. CONCLUSION: A double line septum pellucidum lamina is a normal finding seen in almost 50% of uncomplicated singleton pregnancies. It may be attributed to borders of cell layers within each lamina that form separate specular reflections on both sides; this can be accentuated by ultrasound settings and beam angulation.

Clonal analysis reveals laminar fate multipotency and daughter cell apoptosis of mouse cortical intermediate progenitors.

In developing cerebral cortex, most pyramidal-projection neurons are produced by intermediate progenitors (IPs), derived in turn from radial glial progenitors. Although IPs produce neurons for all cortical layers, it is unknown whether individual IPs produce multiple or single laminar fates, and the potential of IPs for extended proliferation remains uncertain. Previously, we found that, at the population level, early IPs (present during lower-layer neurogenesis) produce lower- and upper-layer neurons, whereas late IPs produce upper-layer neurons only. Here, we employed mosaic analysis with double markers (MADM) in mice to sparsely label early IP clones. Most early IPs produced 1-2 neurons for deep layers only. Less frequently, early IPs produced larger clones (up to 12 neurons) spanning lower and upper layers, or upper layers only. The majority of IP-derived clones (∼66%) were associated with asymmetric cell death after the first division. These data demonstrate that laminar fate is not predetermined, at least in some IPs. Rather, the heterogeneous sizes and laminar fates of early IP clones are correlated with cell division/death/differentiation choices and neuron birthdays, respectively.

The spectrum of brain malformations and disruptions in twins.

Twins have an increased risk for congenital malformations and disruptions, including defects in brain morphogenesis. We analyzed data on brain imaging, zygosity, sex, and fetal demise in 56 proband twins and 7 less affected co-twins with abnormal brain imaging and compared them to population-based data and to a literature series. We separated our series into malformations of cortical development (MCD, N = 39), cerebellar malformations without MCD (N = 13), and brain disruptions (N = 11). The MCD group included 37/39 (95%) with polymicrogyria (PMG), 8/39 (21%) with pia-ependymal clefts (schizencephaly), and 15/39 (38%) with periventricular nodular heterotopia (PNH) including 2 with PNH but not PMG. Cerebellar malformations were found in 19 individuals including 13 with a cerebellar malformation only and another 6 with cerebellar malformation and MCD. The pattern varied from diffuse cerebellar hypoplasia to classic Dandy-Walker malformation. Brain disruptions were seen in 11 individuals with hydranencephaly, porencephaly, or white matter loss without cysts. Our series included an expected statistically significant excess of monozygotic (MZ) twin pairs (22/41 MZ, 54%) compared to population data (482/1448 MZ, 33.3%; p = .0110), and an unexpected statistically significant excess of dizygotic (DZ) twins (19/41, 46%) compared to the literature cohort (1/46 DZ, 2%; p < .0001. Recurrent association with twin-twin transfusion syndrome, intrauterine growth retardation, and other prenatal factors support disruption of vascular perfusion as the most likely unifying cause.

Intermittent Hypoxia Disrupts Adult Neurogenesis and Synaptic Plasticity in the Dentate Gyrus.

Individuals with sleep apnea often exhibit changes in cognitive behaviors consistent with alterations in the hippocampus. It is hypothesized that adult neurogenesis in the dentate gyrus is an ongoing process that maintains normal hippocampal function in many mammalian species, including humans. However, the impact of chronic intermittent hypoxia (IH), a principal consequence of sleep apnea, on hippocampal adult neurogenesis remains unclear. Using a murine model, we examined the impact of 30 d of IH (IH30) on adult neurogenesis and synaptic plasticity in the dentate gyrus. Although IH30 did not affect paired-pulse facilitation, IH30 suppressed long-term potentiation (LTP). Immunohistochemical experiments also indicate that IH perturbs multiple aspects of adult neurogenesis. IH30 increased the number of proliferating Sox2+ neural progenitor cells in the subgranular zone yet reduced the number of doublecortin-positive neurons. Consistent with these findings, cell lineage tracing revealed that IH30 increased the proportion of radial glial cells in the subgranular zone, yet decreased the proportion of adult-born neurons in the dentate gyrus. While administration of a superoxide anion scavenger during IH did not prevent neural progenitor cell proliferation, it mitigated the IH-dependent suppression of LTP and prevented adult-born neuron loss. These data demonstrate that IH causes both reactive oxygen species-dependent and reactive oxygen species-independent effects on adult neurogenesis and synaptic plasticity in the dentate gyrus. Our findings identify cellular and neurophysiological changes in the hippocampus that may contribute to cognitive and behavioral deficits occurring in sleep apnea.SIGNIFICANCE STATEMENT Individuals with sleep apnea experience periods of intermittent hypoxia (IH) that can negatively impact many aspects of brain function. Neurons are continually generated throughout adulthood to support hippocampal physiology and behavior. This study demonstrates that IH exposure attenuates hippocampal long-term potentiation and reduces adult neurogenesis. Antioxidant treatment mitigates these effects indicating that oxidative signaling caused by IH is a significant factor that impairs synaptic plasticity and reduces adult neurogenesis in the hippocampus.

Glial injury in neurotoxicity after pediatric CD19-directed chimeric antigen receptor T cell therapy.

OBJECTIVE: To test whether systemic cytokine release is associated with central nervous system inflammatory responses and glial injury in immune effector cell-associated neurotoxicity syndrome (ICANS) after chimeric antigen receptor (CAR)-T cell therapy in children and young adults. METHODS: We performed a prospective cohort study of clinical manifestations as well as imaging, pathology, CSF, and blood biomarkers on 43 subjects ages 1 to 25 who received CD19-directed CAR/T cells for acute lymphoblastic leukemia (ALL). RESULTS: Neurotoxicity occurred in 19 of 43 (44%) subjects. Nine subjects (21%) had CTCAE grade 3 or 4 neurological symptoms, with no neurotoxicity-related deaths. Reversible delirium, headache, decreased level of consciousness, tremor, and seizures were most commonly observed. Cornell Assessment of Pediatric Delirium (CAPD) scores ≥9 had 94% sensitivity and 33% specificity for grade ≥3 neurotoxicity, and 91% sensitivity and 72% specificity for grade ≥2 neurotoxicity. Neurotoxicity correlated with severity of cytokine release syndrome, abnormal past brain magnetic resonance imaging (MRI), and higher peak CAR-T cell numbers in blood, but not cerebrospinal fluid (CSF). CSF levels of S100 calcium-binding protein B and glial fibrillary acidic protein increased during neurotoxicity, indicating astrocyte injury. There were concomitant increases in CSF white blood cells, protein, interferon-γ (IFNγ), interleukin (IL)-6, IL-10, and granzyme B (GzB), with concurrent elevation of serum IFNγ IL-10, GzB, granulocyte macrophage colony-stimulating factor, macrophage inflammatory protein 1 alpha, and tumor necrosis factor alpha, but not IL-6. We did not find direct evidence of endothelial activation. INTERPRETATION: Our data are most consistent with ICANS as a syndrome of systemic inflammation, which affects the brain through compromise of the neurovascular unit and astrocyte injury. ANN NEUROL 2019.

GSK3ß Inhibition Restores Impaired Neurogenesis in Preterm Neonates With Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) is a common complication of prematurity in infants born at 23-28 weeks of gestation. Survivors exhibit impaired growth of the cerebral cortex and neurodevelopmental sequeale, but the underlying mechanism(s) are obscure. Previously, we have shown that neocortical neurogenesis continues until at least 28 gestational weeks. This renders the prematurely born infants vulnerable to impaired neurogenesis. Here, we hypothesized that neurogenesis is impaired by IVH, and that signaling through GSK3ß, a critical intracellular kinase regulated by Wnt and other pathways, mediates this effect. These hypotheses were tested observationally in autopsy specimens from premature infants, and experimentally in a premature rabbit IVH model. Significantly, in premature infants with IVH, the number of neurogenic cortical progenitor cells was reduced compared with infants without IVH, indicating acutely decreased neurogenesis. This finding was corroborated in the rabbit IVH model, which further demonstrated reduction of upper layer cortical neurons after longer survival. Both the acute reduction of neurogenic progenitors, and the subsequent decrease of upper layer neurons, were rescued by treatment with AR-A014418, a specific inhibitor of GSK3ß. Together, these results indicate that IVH impairs late stages of cortical neurogenesis, and suggest that treatment with GSK3ß inhibitors may enhance neurodevelopment in premature infants with IVH.

Intermediate progenitors and Tbr2 in cortical development.

In developing cerebral cortex, intermediate progenitors (IPs) are transit amplifying cells that specifically express Tbr2 (gene: Eomes), a T-box transcription factor. IPs are derived from radial glia (RG) progenitors, the neural stem cells of developing cortex. In turn, IPs generate glutamatergic projection neurons (PNs) exclusively. IPs are found in ventricular and subventricular zones, where they differentiate as distinct ventricular IP (vIP) and outer IP (oIP) subtypes. Morphologically, IPs have short processes, resembling filopodia or neurites, that transiently contact other cells, most importantly dividing RG cells to mediate Delta-Notch signaling. Also, IPs secrete a chemokine, Cxcl12, which guides interneuron and microglia migrations and promotes thalamocortical axon growth. In mice, IPs produce clones of 1-12 PNs, sometimes spanning multiple layers. After mitosis, IP daughter cells undergo asymmetric cell death in the majority of instances. In mice, Tbr2 is necessary for PN differentiation and subtype specification, and to repress IP-genic transcription factors. Tbr2 directly represses Insm1, an IP-genic transcription factor gene, as well as Pax6, a key activator of Tbr2 transcription. Without Tbr2, abnormal IPs transiently accumulate in elevated numbers. More broadly, Tbr2 regulates the transcriptome by activating or repressing hundreds of direct target genes. Notably, Tbr2 'unlocks' and activates PN-specific genes, such as Tbr1, by recruiting Jmjd3, a histone H3K27me3 demethylase that removes repressive epigenetic marks placed by polycomb repressive complex 2. IPs have played an important role in the evolution and gyrification of mammalian cerebral cortex, and TBR2 is essential for human brain development.

New insights into the development of the human cerebral cortex.

The cerebral cortex constitutes more than half the volume of the human brain and is presumed to be responsible for the neuronal computations underlying complex phenomena, such as perception, thought, language, attention, episodic memory and voluntary movement. Rodent models are extremely valuable for the investigation of brain development, but cannot provide insight into aspects that are unique or highly derived in humans. Many human psychiatric and neurological conditions have developmental origins but cannot be studied adequately in animal models. The human cerebral cortex has some unique genetic, molecular, cellular and anatomical features, which need to be further explored. The Anatomical Society devoted its summer meeting to the topic of Human Brain Development in June 2018 to tackle these important issues. The meeting was organized by Gavin Clowry (Newcastle University) and Zoltán Molnár (University of Oxford), and held at St John's College, Oxford. The participants provided a broad overview of the structure of the human brain in the context of scaling relationships across the brains of mammals, conserved principles and recent changes in the human lineage. Speakers considered how neuronal progenitors diversified in human to generate an increasing variety of cortical neurons. The formation of the earliest cortical circuits of the earliest generated neurons in the subplate was discussed together with their involvement in neurodevelopmental pathologies. Gene expression networks and susceptibility genes associated to neurodevelopmental diseases were discussed and compared with the networks that can be identified in organoids developed from induced pluripotent stem cells that recapitulate some aspects of in vivo development. New views were discussed on the specification of glutamatergic pyramidal and γ-aminobutyric acid (GABA)ergic interneurons. With the advancement of various in vivo imaging methods, the histopathological observations can be now linked to in vivo normal conditions and to various diseases. Our review gives a general evaluation of the exciting new developments in these areas. The human cortex has a much enlarged association cortex with greater interconnectivity of cortical areas with each other and with an expanded thalamus. The human cortex has relative enlargement of the upper layers, enhanced diversity and function of inhibitory interneurons and a highly expanded transient subplate layer during development. Here we highlight recent studies that address how these differences emerge during development focusing on diverse facets of our evolution.

Growth and folding of the mammalian cerebral cortex: from molecules to malformations.

The size and extent of folding of the mammalian cerebral cortex are important factors that influence a species' cognitive abilities and sensorimotor skills. Studies in various animal models and in humans have provided insight into the mechanisms that regulate cortical growth and folding. Both protein-coding genes and microRNAs control cortical size, and recent progress in characterizing basal progenitor cells and the genes that regulate their proliferation has contributed to our understanding of cortical folding. Neurological disorders linked to disruptions in cortical growth and folding have been associated with novel neurogenetic mechanisms and aberrant signalling pathways, and these findings have changed concepts of brain evolution and may lead to new medical treatments for certain disorders.

Prenatal and early life diesel exhaust exposure disrupts cortical lamina organization: Evidence for a reelin-related pathogenic pathway induced by interleukin-6.

Several epidemiological studies have shown associations between developmental exposure to traffic-related air pollution and increased risk for autism spectrum disorders (ASD), a spectrum of neurodevelopmental disorders with increasing prevalence rate in the United States. Though animal studies have provided support for these associations, little is known regarding possible underlying mechanisms. In a previous study we found that exposure of C57BL/6J mice of both sexes to environmentally relevant levels (250-300 µg/m3) of diesel exhaust (DE) from embryonic day 0 to postnatal day 21 (E0 to PND21) caused significant changes in all three characteristic behavioral domains of ASD in the offspring. In the present study we investigated a potential mechanistic pathway that may be of relevance for ASD-like changes associated with developmental DE exposure. Using the same DE exposure protocol (250-300 µg/m3 DE from E0 to PND21) several molecular markers were examined in the brains of male and female mice at PND3, 21, and 60. Exposure to DE as above increased levels of interleukin-6 (IL-6) in placenta and in neonatal brain. The JAK2/STAT3 pathway, a target for IL-6, was activated by STAT3 phosphorylation, and the expression of DNA methyltransferase 1 (DNMT1), a STAT3 target gene, was increased in DE-exposed neonatal brain. DNMT1 has been reported to down-regulate expression of reelin (RELN), an extracellular matrix glycoprotein important in regulating the processes of neuronal migration. RELN is considered an important modulator for ASD, since there are several polymorphisms in this gene linked to the disease, and since lower levels of RELN have been reported in brains of ASD patients. We observed decreased RELN expression in brains of the DE-exposed mice at PND3. Since disorganized patches in the prefrontal cortex have been reported in ASD patients and disrupted cortical organization has been found in RELN-deficient mice, we also assessed cortical organization, by labeling cells expressing the lamina-specific-markers RELN and calretinin. In DE-exposed mice we found increased cell density in deeper cortex (lamina layers VI-IV) for cells expressing either RELN or calretinin. These findings demonstrate that developmental DE exposure is associated with subtle disorganization of the cerebral cortex at PND60, and suggest a pathway involving IL-6, STAT3, and DNMT1 leading to downregulation of RELN expression that could be contributing to this long-lasting disruption in cortical laminar organization.

C-Terminal Region Truncation of RELN Disrupts an Interaction with VLDLR, Causing Abnormal Development of the Cerebral Cortex and Hippocampus.

We discovered a hypomorphic reelin (Reln) mutant with abnormal cortical lamination and no cerebellar hypoplasia. This mutant, RelnCTRdel, carries a chemically induced splice-site mutation that truncates the C-terminal region (CTR) domain of RELN protein and displays remarkably distinct phenotypes from reeler The mutant does not have an inverted cortex, but cortical neurons overmigrate and invade the marginal zone, which are characteristics similar to a phenotype seen in the cerebral cortex of Vldlrnull mice. The dentate gyrus shows a novel phenotype: the infrapyramidal blade is absent, while the suprapyramidal blade is present and laminated. Genetic epistasis analysis showed that RelnCTRdel/Apoer2null double homozygotes have phenotypes akin to those of reeler mutants, while RelnCTRdel/Vldlrnull mice do not. Given that the receptor double knock-out mice resemble reeler mutants, we infer that RelnCTRdel/Apoer2null double homozygotes have both receptor pathways disrupted. This suggests that CTR-truncation disrupts an interaction with VLDLR (very low-density lipoprotein receptor), while the APOER2 signaling pathway remains active, which accounts for the hypomorphic phenotype in RelnCTRdel mice. A RELN-binding assay confirms that CTR truncation significantly decreases RELN binding to VLDLR, but not to APOER2. Together, the in vitro and in vivo results demonstrate that the CTR domain confers receptor-binding specificity of RELN. SIGNIFICANCE STATEMENT: Reelin signaling is important for brain development and is associated with human type II lissencephaly. Reln mutations in mice and humans are usually associated with cerebellar hypoplasia. A new Reln mutant with a truncation of the C-terminal region (CTR) domain shows that Reln mutation can cause abnormal phenotypes in the cortex and hippocampus without cerebellar hypoplasia. Genetic analysis suggested that CTR truncation disrupts an interaction with the RELN receptor VLDLR (very low-density lipoprotein receptor); this was confirmed by a RELN-binding assay. This result provides a mechanistic explanation for the hypomorphic phenotype of the CTR-deletion mutant, and further suggests that Reln mutations may cause more subtle forms of human brain malformation than classic lissencephalies.

Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly.

Mutations of genes within the phosphatidylinositol-3-kinase (PI3K)-AKT-MTOR pathway are well known causes of brain overgrowth (megalencephaly) as well as segmental cortical dysplasia (such as hemimegalencephaly, focal cortical dysplasia and polymicrogyria). Mutations of the AKT3 gene have been reported in a few individuals with brain malformations, to date. Therefore, our understanding regarding the clinical and molecular spectrum associated with mutations of this critical gene is limited, with no clear genotype-phenotype correlations. We sought to further delineate this spectrum, study levels of mosaicism and identify genotype-phenotype correlations of AKT3-related disorders. We performed targeted sequencing of AKT3 on individuals with these phenotypes by molecular inversion probes and/or Sanger sequencing to determine the type and level of mosaicism of mutations. We analysed all clinical and brain imaging data of mutation-positive individuals including neuropathological analysis in one instance. We performed ex vivo kinase assays on AKT3 engineered with the patient mutations and examined the phospholipid binding profile of pleckstrin homology domain localizing mutations. We identified 14 new individuals with AKT3 mutations with several phenotypes dependent on the type of mutation and level of mosaicism. Our comprehensive clinical characterization, and review of all previously published patients, broadly segregates individuals with AKT3 mutations into two groups: patients with highly asymmetric cortical dysplasia caused by the common p.E17K mutation, and patients with constitutional AKT3 mutations exhibiting more variable phenotypes including bilateral cortical malformations, polymicrogyria, periventricular nodular heterotopia and diffuse megalencephaly without cortical dysplasia. All mutations increased kinase activity, and pleckstrin homology domain mutants exhibited enhanced phospholipid binding. Overall, our study shows that activating mutations of the critical AKT3 gene are associated with a wide spectrum of brain involvement ranging from focal or segmental brain malformations (such as hemimegalencephaly and polymicrogyria) predominantly due to mosaic AKT3 mutations, to diffuse bilateral cortical malformations, megalencephaly and heterotopia due to constitutional AKT3 mutations. We also provide the first detailed neuropathological examination of a child with extreme megalencephaly due to a constitutional AKT3 mutation. This child has one of the largest documented paediatric brain sizes, to our knowledge. Finally, our data show that constitutional AKT3 mutations are associated with megalencephaly, with or without autism, similar to PTEN-related disorders. Recognition of this broad clinical and molecular spectrum of AKT3 mutations is important for providing early diagnosis and appropriate management of affected individuals, and will facilitate targeted design of future human clinical trials using PI3K-AKT pathway inhibitors.

A dual-fluorescence reporter in the Eomes locus for live imaging and medium-term lineage tracing.

The T-box transcription factor Eomes (also known as Tbr2) shows short-lived expression in various localized domains of the embryo, including epiblast cells during gastrulation and intermediate progenitor cells in the cerebral cortex. In these tissues Eomes fulfills crucial roles for lineage specification of progenitors. To directly observe Eomes-dependent cell lineages in the living embryo, we generated a novel dual-fluorescence reporter allele that expresses a membrane-bound tdTomato protein for investigation of cell morphology and a nuclear GFP for cell tracing. This allele recapitulates endogenous EOMES protein expression and is suitable for live imaging. We found that the allele can also be used as a short-to-medium-term lineage tracer, as GFP persists in cells longer than EOMES protein and marks Eomes-dependent lineages with a timeframe of days to weeks depending on the proliferation rate. In summary, we present a novel genetic tool for investigation of Eomes-dependent cell types by live imaging and lineage tracing.

PI3K/AKT pathway mutations cause a spectrum of brain malformations from megalencephaly to focal cortical dysplasia.

Malformations of cortical development containing dysplastic neuronal and glial elements, including hemimegalencephaly and focal cortical dysplasia, are common causes of intractable paediatric epilepsy. In this study we performed multiplex targeted sequencing of 10 genes in the PI3K/AKT pathway on brain tissue from 33 children who underwent surgical resection of dysplastic cortex for the treatment of intractable epilepsy. Sequencing results were correlated with clinical, imaging, pathological and immunohistological phenotypes. We identified mosaic activating mutations in PIK3CA and AKT3 in this cohort, including cancer-associated hotspot PIK3CA mutations in dysplastic megalencephaly, hemimegalencephaly, and focal cortical dysplasia type IIa. In addition, a germline PTEN mutation was identified in a male with hemimegalencephaly but no peripheral manifestations of the PTEN hamartoma tumour syndrome. A spectrum of clinical, imaging and pathological abnormalities was found in this cohort. While patients with more severe brain imaging abnormalities and systemic manifestations were more likely to have detected mutations, routine histopathological studies did not predict mutation status. In addition, elevated levels of phosphorylated S6 ribosomal protein were identified in both neurons and astrocytes of all hemimegalencephaly and focal cortical dysplasia type II specimens, regardless of the presence or absence of detected PI3K/AKT pathway mutations. In contrast, expression patterns of the T308 and S473 phosphorylated forms of AKT and in vitro AKT kinase activities discriminated between mutation-positive dysplasia cortex, mutation-negative dysplasia cortex, and non-dysplasia epilepsy cortex. Our findings identify PI3K/AKT pathway mutations as an important cause of epileptogenic brain malformations and establish megalencephaly, hemimegalencephaly, and focal cortical dysplasia as part of a single pathogenic spectrum.

The protomap is propagated to cortical plate neurons through an Eomes-dependent intermediate map.

The cortical area map is initially patterned by transcription factor (TF) gradients in the neocortical primordium, which define a "protomap" in the embryonic ventricular zone (VZ). However, mechanisms that propagate regional identity from VZ progenitors to cortical plate (CP) neurons are unknown. Here we show that the VZ, subventricular zone (SVZ), and CP contain distinct molecular maps of regional identity, reflecting different gene expression gradients in radial glia progenitors, intermediate progenitors, and projection neurons, respectively. The "intermediate map" in the SVZ is modulated by Eomes (also known as Tbr2), a T-box TF. Eomes inactivation caused rostrocaudal shifts in SVZ and CP gene expression, with loss of corticospinal axons and gain of corticotectal projections. These findings suggest that cortical areas and connections are shaped by sequential maps of regional identity, propagated by the Pax6 → Eomes → Tbr1 TF cascade. In humans, PAX6, EOMES, and TBR1 have been linked to intellectual disability and autism.

Genetic elimination of GABAergic neurotransmission reveals two distinct pacemakers for spontaneous waves of activity in the developing mouse cortex.

Many structures of the mammalian CNS generate propagating waves of electrical activity early in development. These waves are essential to CNS development, mediating a variety of developmental processes, such as axonal outgrowth and pathfinding, synaptogenesis, and the maturation of ion channel and receptor properties. In the mouse cerebral cortex, waves of activity occur between embryonic day 18 and postnatal day 8 and originate in pacemaker circuits in the septal nucleus and the piriform cortex. Here we show that genetic knock-out of the major synthetic enzyme for GABA, GAD67, selectively eliminates the picrotoxin-sensitive fraction of these waves. The waves that remain in the GAD67 knock-out have a much higher probability of propagating into the dorsal neocortex, as do the picrotoxin-resistant fraction of waves in controls. Field potential recordings at the point of wave initiation reveal different electrical signatures for GABAergic and glutamatergic waves. These data indicate that: (1) there are separate GABAergic and glutamatergic pacemaker circuits within the piriform cortex, each of which can initiate waves of activity; (2) the glutamatergic pacemaker initiates waves that preferentially propagate into the neocortex; and (3) the initial appearance of the glutamatergic pacemaker does not require preceding GABAergic waves. In the absence of GAD67, the electrical activity underlying glutamatergic waves shows greatly increased tendency to burst, indicating that GABAergic inputs inhibit the glutamatergic pacemaker, even at stages when GABAergic pacemaker circuitry can itself initiate waves.