Mutations that cause neurological phenotypes are highly informative with regard to mechanisms governing human brain function and disease. We report autosomal recessive mutations in the enzyme glutamate pyruvate transaminase 2 (GPT2) in large kindreds initially ascertained for intellectual and developmental disability (IDD). GPT2 [also known as alanine transaminase 2 (ALT2)] is one of two related transaminases that catalyze the reversible addition of an amino group from glutamate to pyruvate, yielding alanine and α-ketoglutarate. In addition to IDD, all affected individuals show postnatal microcephaly and ∼80% of those followed over time show progressive motor symptoms, a spastic paraplegia. Homozygous nonsense p.Arg404* and missense p.Pro272Leu mutations are shown biochemically to be loss of function. The GPT2 gene demonstrates increasing expression in brain in the early postnatal period, and GPT2 protein localizes to mitochondria. Akin to the human phenotype, Gpt2-null mice exhibit reduced brain growth. Through metabolomics and direct isotope tracing experiments, we find a number of metabolic abnormalities associated with loss of Gpt2. These include defects in amino acid metabolism such as low alanine levels and elevated essential amino acids. Also, we find defects in anaplerosis, the metabolic process involved in replenishing TCA cycle intermediates. Finally, mutant brains demonstrate misregulated metabolites in pathways implicated in neuroprotective mechanisms previously associated with neurodegenerative disorders. Overall, our data reveal an important role for the GPT2 enzyme in mitochondrial metabolism with relevance to developmental as well as potentially to neurodegenerative mechanisms.

The molecular mechanisms controlling aqueous humor (AQH) outflow and IOP need much further definition. The mouse is a powerful system for characterizing the mechanistic basis of AQH outflow. To enhance outflow studies in mice, we developed a perfusion system that is based on human anterior chamber perfusion culture systems. Our mouse system permits previously impractical experiments.

Improved understanding of how the human brain is "wired" on a macroscale may now be possible due to the emerging field of MRI connectomics. However, mapping the rapidly developing infant brain networks poses challenges. In this study, we applied an automated template-free "baby connectome" framework using diffusion MRI to non-invasively map the structural brain networks in subjects of different ages, including premature neonates, term-born neonates, six-month-old infants, and adults. We observed increasing brain network integration and decreasing segregation with age in term-born subjects. We also explored how the equal area nodes can be grouped into modules without any prior anatomical information--an important step toward a fully network-driven registration and analysis of brain connectivity.

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.

Here, we use a mouse model (DBA/2J) to readdress the location of insult(s) to retinal ganglion cells (RGCs) in glaucoma. We localize an early sign of axon damage to an astrocyte-rich region of the optic nerve just posterior to the retina, analogous to the lamina cribrosa. In this region, a network of astrocytes associates intimately with RGC axons. Using BAX-deficient DBA/2J mice, which retain all of their RGCs, we provide experimental evidence for an insult within or very close to the lamina in the optic nerve. We show that proximal axon segments attached to their cell bodies survive to the proximity of the lamina. In contrast, axon segments in the lamina and behind the eye degenerate. Finally, the Wld(s) allele, which is known to protect against insults to axons, strongly protects against DBA/2J glaucoma and preserves RGC activity as measured by pattern electroretinography. These experiments provide strong evidence for a local insult to axons in the optic nerve.

Destrin (DSTN) is a member of the ADF/cofilin family of proteins and is an important regulator of actin dynamics. The primary function of destrin is to depolymerize filamentous actin into its monomeric form and promote filament severing. While progress has been made in understanding the biochemical functions of the ADF/cofilin proteins, the study of an animal model for cells deficient for DSTN provides an opportunity to investigate the physiological processes regulated by proper actin dynamics in vivo. A spontaneous mouse mutant, corneal disease 1(corn1), is deficient for DSTN, which causes epithelial hyperproliferation and neovascularization in the cornea. Dstn(corn1) mice exhibit an actin dynamics defect in the cornea as evidenced by the formation of actin stress fibers in the epithelial cells. Previously, we observed a significant infiltration of leukocytes into the cornea of Dstn(corn1) mice as well as the upregulation of proinflammatory molecules. In this study, we sought to characterize this inflammatory condition and explore the physiological mechanism through which a loss of Dstn function leads to inflammation.

The human cerebral cortex is distinguished by its large size and abundant gyrification, or folding. However, the evolutionary mechanisms that drive cortical size and structure are unknown. Although genes that are essential for cortical developmental expansion have been identified from the genetics of human primary microcephaly (a disorder associated with reduced brain size and intellectual disability) 1 , studies of these genes in mice, which have a smooth cortex that is one thousand times smaller than the cortex of humans, have provided limited insight. Mutations in abnormal spindle-like microcephaly-associated (ASPM), the most common recessive microcephaly gene, reduce cortical volume by at least 50% in humans2-4, but have little effect on the brains of mice5-9; this probably reflects evolutionarily divergent functions of ASPM10,11. Here we used genome editing to create a germline knockout of Aspm in the ferret (Mustela putorius furo), a species with a larger, gyrified cortex and greater neural progenitor cell diversity12-14 than mice, and closer protein sequence homology to the human ASPM protein. Aspm knockout ferrets exhibit severe microcephaly (25-40% decreases in brain weight), reflecting reduced cortical surface area without significant change in cortical thickness, as has been found in human patients3,4, suggesting that loss of 'cortical units' has occurred. The cortex of fetal Aspm knockout ferrets displays a very large premature displacement of ventricular radial glial cells to the outer subventricular zone, where many resemble outer radial glia, a subtype of neural progenitor cells that are essentially absent in mice and have been implicated in cerebral cortical expansion in primates12-16. These data suggest an evolutionary mechanism by which ASPM regulates cortical expansion by controlling the affinity of ventricular radial glial cells for the ventricular surface, thus modulating the ratio of ventricular radial glial cells, the most undifferentiated cell type, to outer radial glia, a more differentiated progenitor.

The shape and function of plant cells are often highly interdependent. The puzzle-shaped cells that appear in the epidermis of many plants are a striking example of a complex cell shape, however their functional benefit has remained elusive. We propose that these intricate forms provide an effective strategy to reduce mechanical stress in the cell wall of the epidermis. When tissue-level growth is isotropic, we hypothesize that lobes emerge at the cellular level to prevent formation of large isodiametric cells that would bulge under the stress produced by turgor pressure. Data from various plant organs and species support the relationship between lobes and growth isotropy, which we test with mutants where growth direction is perturbed. Using simulation models we show that a mechanism actively regulating cellular stress plausibly reproduces the development of epidermal cell shape. Together, our results suggest that mechanical stress is a key driver of cell-shape morphogenesis.

Malformations of cortical development (MCDs) compose a diverse range of disorders that are common causes of neurodevelopmental delay and epilepsy. With improved imaging and genetic methodologies, the underlying molecular and pathobiological characteristics of several MCDs have been recently elucidated. In this review, we discuss genetic and molecular alterations that disrupt normal cortical development, with emphasis on recent discoveries, and provide detailed radiological features of the most common and important MCDs. Ann Neurol 2016;80:797-810.

Organ size and shape are precisely regulated to ensure proper function. The four sepals in each Arabidopsis thaliana flower must maintain the same size throughout their growth to continuously enclose and protect the developing bud. Here we show that DEVELOPMENT RELATED MYB-LIKE 1 (DRMY1) is required for both timing of organ initiation and proper growth, leading to robust sepal size in Arabidopsis. Within each drmy1 flower, the initiation of some sepals is variably delayed. Late-initiating sepals in drmy1 mutants remain smaller throughout development, resulting in variability in sepal size. DRMY1 focuses the spatiotemporal signalling patterns of the plant hormones auxin and cytokinin, which jointly control the timing of sepal initiation. Our findings demonstrate that timing of organ initiation, together with growth and maturation, contribute to robust organ size.

Mendelian and early-onset severe psychiatric phenotypes often involve genetic variants having a large effect, offering opportunities for genetic discoveries and early therapeutic interventions. Here, the index case is an 18-year-old boy, who at 14 years of age had a decline in cognitive functioning over the course of a year and subsequently presented with catatonia, auditory and visual hallucinations, paranoia, aggression, mood dysregulation, and disorganized thoughts. Exome sequencing revealed a stop-gain mutation in RCL1 (NM_005772.4:c.370 C > T, p.Gln124Ter), encoding an RNA 3'-terminal phosphate cyclase-like protein that is highly conserved across eukaryotic species. Subsequent investigations across two academic medical centers identified eleven additional cases of RCL1 copy number variations (CNVs) with varying neurodevelopmental or psychiatric phenotypes. These findings suggest that dosage variation of RCL1 contributes to a range of neurological and clinical phenotypes.

Background Impairments in fetal oxygen delivery have been implicated in brain dysmaturation seen in congenital heart disease (CHD), suggesting a role for in utero transplacental oxygen therapy. We applied a novel imaging tool to quantify fetal cerebral oxygenation by measuring T2* decay. We compared T2* in fetuses with CHD with controls with a focus on cardiovascular physiologies (transposition or left-sided obstruction) and described the effect of brief administration of maternal hyperoxia on T2* decay. Methods and Results This is a prospective study performed on pregnant mothers with a prenatal diagnosis of CHD compared with controls in the third trimester. Participants underwent a fetal brain magnetic resonance imaging scan including a T2* sequence before and after maternal hyperoxia. Comparisons were made between control and CHD fetuses including subgroup analyses by cardiac physiology. Forty-four mothers (CHD=24, control=20) participated. Fetuses with CHD had lower total brain volume (238.2 mm3, 95% CI, 224.6-251.9) compared with controls (262.4 mm3, 95% CI, 245.0-279.8, P=0.04). T2* decay time was faster in CHD compared with controls (beta=-14.4, 95% CI, -23.3 to -5.6, P=0.002). The magnitude of change in T2* with maternal hyperoxia was higher in fetuses with transposition compared with controls (increase of 8.4 ms, 95% CI, 0.5-14.3, P=0.01), though between-subject variability was noted. Conclusions Cerebral tissue oxygenation is lower in fetuses with complex CHD. There was variability in the response to maternal hyperoxia by CHD subgroup that can be tested in future larger studies. Cardiovascular physiology is critical when designing neuroprotective clinical trials in the fetus with CHD.

Morphogenesis emerges from complex multiscale interactions between genetic and mechanical processes. To understand these processes, the evolution of cell shape, proliferation and gene expression must be quantified. This quantification is usually performed either in full 3D, which is computationally expensive and technically challenging, or on 2D planar projections, which introduces geometrical artifacts on highly curved organs. Here we present MorphoGraphX ( www.MorphoGraphX.org), a software that bridges this gap by working directly with curved surface images extracted from 3D data. In addition to traditional 3D image analysis, we have developed algorithms to operate on curved surfaces, such as cell segmentation, lineage tracking and fluorescence signal quantification. The software's modular design makes it easy to include existing libraries, or to implement new algorithms. Cell geometries extracted with MorphoGraphX can be exported and used as templates for simulation models, providing a powerful platform to investigate the interactions between shape, genes and growth.

Cerebral cortical folding becomes dramatically more complex in the fetal brain during the 3rd trimester of gestation; the process continues in a similar fashion in children who are born prematurely. To quantify this morphological development, it is necessary to extract the interface between gray matter and white matter, which is particularly challenging due to changing tissue contrast during brain maturation. We employed the well-established CIVET pipeline to extract this cortical surface, with point correspondence across subjects, using a surface-based spherical registration. We then developed a variant of the pipeline, called NEOCIVET, that quantified cortical folding using mean curvature and sulcal depth while addressing the well-known problems of poor and temporally-varying gray/white contrast as well as motion artifact in neonatal MRI. NEOCIVET includes: i) a tissue classification technique that analyzed multi-atlas texture patches using the nonlocal mean estimator and subsequently applied a label fusion approach based on a joint probability between templates, ii) neonatal template construction based on age-specific sub-groups, and iii) masking of non-interesting structures using label-fusion approaches. These techniques replaced modules that might be suboptimal for regional analysis of poor-contrast neonatal cortex. The proposed segmentation method showed more accurate results in subjects with various ages and with various degrees of motion compared to state-of-the-art methods. In the analysis of 158 preterm-born neonates, many with multiple scans (n=231; 26-40weeks postmenstrual age at scan), NEOCIVET identified increases in cortical folding over time in numerous cortical regions (mean curvature: +0.003/week; sulcal depth: +0.04mm/week) while folding did not change in major sulci that are known to develop early (corrected p<0.05). The proposed pipeline successfully mapped cortical structural development, supporting current models of cerebral morphogenesis, and furthermore, revealed impairment of cortical folding in extremely preterm newborns relative to relatively late preterm newborns, demonstrating its potential to provide biomarkers of prematurity-related developmental outcome.

Neuronal loss and axonal degeneration are important pathological features of many neurodegenerative diseases. The molecular mechanisms underlying the majority of axonal degeneration conditions remain unknown. To better understand axonal degeneration, we studied a mouse mutant wabbler-lethal (wl). Wabbler-lethal (wl) mutant mice develop progressive ataxia with pronounced neurodegeneration in the central and peripheral nervous system. Previous studies have led to a debate as to whether myelinopathy or axonopathy is the primary cause of neurodegeneration observed in wl mice. Here we provide clear evidence that wabbler-lethal mutants develop an axonopathy, and that this axonopathy is modulated by Wld(s) and Bax mutations. In addition, we have identified the gene harboring the disease-causing mutations as Atp8a2. We studied three wl alleles and found that all result from mutations in the Atp8a2 gene. Our analysis shows that ATP8A2 possesses phosphatidylserine translocase activity and is involved in localization of phosphatidylserine to the inner leaflet of the plasma membrane. Atp8a2 is widely expressed in the brain, spinal cord, and retina. We assessed two of the mutant alleles of Atp8a2 and found they are both nonfunctional for the phosphatidylserine translocase activity. Thus, our data demonstrate for the first time that mutation of a mammalian phosphatidylserine translocase causes axon degeneration and neurodegenerative disease.

Pigment dispersion syndrome causes iris pigment release and often progresses to elevated intraocular pressure and pigmentary glaucoma (PG). Because melanin pigment can have adjuvant like properties and because the Gpnmb gene, which contributes to pigment dispersion in DBA/2J (D2) mice, is expressed in dendritic cells, we tested the hypothesis that ocular immune abnormalities participate in PG phenotypes. Strikingly, we show that D2 eyes exhibit defects of the normally immunosuppressive ocular microenvironment including inability of aqueous humor to inhibit T cell activation, failure to support anterior chamber (AC)-associated immune deviation, and loss of ocular immune privilege. Histologic analysis demonstrates infiltration of inflammatory leukocytes into the AC and their accumulation within the iris, whereas clinical indications of inflammation are typically very mild to undetectable. Importantly, some of these abnormalities precede clinical indications of pigment dispersal, suggesting an early role in disease etiology. Using bone marrow chimeras, we show that lymphohematopoietic cell lineages largely dictate the progression of pigment dispersion, the ability of the eye to support induction of AC-associated immune deviation, and the integrity of the blood/ocular barrier. These results suggest previously unsuspected roles for bone marrow-derived cells and ocular immune privilege in the pathogenesis of PG.

Genes associated with human microcephaly, a condition characterized by a small brain, include critical regulators of proliferation, cell fate and DNA repair. We describe a syndrome of congenital microcephaly and diverse defects in cerebral cortical architecture. Genome-wide linkage analysis in two families identified a 7.5-Mb locus on chromosome 19q13.12 containing 148 genes. Targeted high throughput sequence analysis of linked genes in each family yielded > 4,000 DNA variants and implicated a single gene, WDR62, as harboring potentially deleterious changes. We subsequently identified additional WDR62 mutations in four other families. Magnetic resonance imaging and postmortem brain analysis supports important roles for WDR62 in the proliferation and migration of neuronal precursors. WDR62 is a WD40 repeat-containing protein expressed in neuronal precursors as well as in postmitotic neurons in the developing brain and localizes to the spindle poles of dividing cells. The diverse phenotypes of WDR62 suggest it has central roles in many aspects of cerebral cortical development.

Schizencephaly is a malformation of cortical development characterized by gray matter-lined clefts in the cerebral cortex and a range of neurological presentations. In some cases, there are features of septo-optic dysplasia concurrently with schizencephaly. The etiologies of both schizencephaly and septo-optic dysplasia are thought to be heterogeneous, but there is evidence that at least some cases have genetic origin. We hypothesized that these disorders may be caused by mutations in three candidate genes: LHX2, a gene with an important cortical patterning role, and HESX1 and SOX2, genes that have been associated with septo-optic dysplasia. We sequenced a large cohort of patients with schizencephaly, some with features of septo-optic dysplasia, for mutations in these genes. No pathogenic mutations were observed, suggesting that other genes or non-genetic factors influencing genes critical to brain development must be responsible for schizencephaly.

Defining the structural and functional connectivity of the human brain (the human "connectome") is a basic challenge in neuroscience. Recently, techniques for noninvasively characterizing structural connectivity networks in the adult brain have been developed using diffusion and high-resolution anatomic MRI. The purpose of this study was to establish a framework for assessing structural connectivity in the newborn brain at any stage of development and to show how network properties can be derived in a clinical cohort of six-month old infants sustaining perinatal hypoxic ischemic encephalopathy (HIE). Two different anatomically unconstrained parcellation schemes were proposed and the resulting network metrics were correlated with neurological outcome at 6 months. Elimination and correction of unreliable data, automated parcellation of the cortical surface, and assembling the large-scale baby connectome allowed an unbiased study of the network properties of the newborn brain using graph theoretic analysis. In the application to infants with HIE, a trend to declining brain network integration and segregation was observed with increasing neuromotor deficit scores.

An electroretinogram (ERG) screen identified a mouse with a normal a-wave but lacking a b-wave, and as such it was designated no b-wave3 (nob3). The nob3 phenotype mapped to chromosome 11 in a region containing the metabotropic glutamate receptor 6 gene (Grm6). Sequence analyses of cDNA identified a splicing error in Grm6, introducing an insertion and an early stop codon into the mRNA of affected mice (designated Grm6(nob3)). Immunohistochemistry of the Grm6(nob3) retina showed that GRM6 was absent. The ERG and visual behaviour abnormalities of Grm6(nob3) mice are similar to Grm6(nob4) animals, and similar deficits were seen in compound heterozygotes (Grm6(nob4/nob3)), indicating that Grm6(nob3) is allelic to Grm6(nob4). Visual responses of Grm6(nob3) retinal ganglion cells (RGCs) to light onset were abnormal. Grm6(nob3) ON RGCs were rarely recorded, but when they were, had ill-defined receptive field (RF) centres and delayed onset latencies. When Grm6(nob3) OFF-centre RGC responses were evoked by full-field stimulation, significantly fewer converted that response to OFF/ON compared to Grm6(nob4) RGCs. Grm6(nob4/nob3) RGC responses verified the conclusion that the two mutants are allelic. We propose that Grm6(nob3) is a new model of human autosomal recessive congenital stationary night blindness. However, an allelic difference between Grm6(nob3) and Grm6(nob4) creates a disparity in inner retinal processing. Because the localization of GRM6 is limited to bipolar cells in the On pathway, the observed difference between RGCs in these mutants is likely to arise from differences in their inputs.