Matthew C. Judson

Angelman syndrome: insights into genomic imprinting and neurodevelopmental phenotypes

Angelman syndrome (AS) is a severe genetic disorder caused by mutations or deletions of the maternally inherited UBE3A gene. UBE3A encodes an E3 ubiquitin ligase that is expressed biallelically in most tissues but is maternally expressed in almost all neurons. In this review, we describe recent advances in understanding the expression and function of UBE3A in the brain and the etiology of AS. We highlight current AS model systems, epigenetic mechanisms of UBE3A regulation, and the identification of potential UBE3A substrates in the brain. In the process, we identify major gaps in our knowledge that, if bridged, could move us closer to identifying treatments for this debilitating neurodevelopmental disorder.

Dynamic gene and protein expression patterns of the autism-associated met receptor tyrosine kinase in the developing mouse forebrain

The establishment of appropriate neural circuitry depends on the coordination of multiple developmental events across space and time. These events include proliferation, migration, differentiation, and survival—all of which can be mediated by hepatocyte growth factor (HGF) signaling through the Met receptor tyrosine kinase. We previously found a functional promoter variant of the MET gene to be associated with autism spectrum disorder, suggesting that forebrain circuits governing social and emotional function may be especially vulnerable to developmental disruptions in HGF/Met signaling. However, little is known about the spatiotemporal distribution of Met expression in the forebrain during the development of such circuits. To advance our understanding of the neurodevelopmental influences of Met activation, we employed complementary Western blotting, in situ hybridization, and immunohistochemistry to comprehensively map Met transcript and protein expression throughout perinatal and postnatal development of the mouse forebrain. Our studies reveal complex and dynamic spatiotemporal patterns of expression during this period. Spatially, Met transcript is localized primarily to specific populations of projection neurons within the neocortex and in structures of the limbic system, including the amygdala, hippocampus, and septum. Met protein appears to be principally located in axon tracts. Temporally, peak expression of transcript and protein occurs during the second postnatal week. This period is characterized by extensive neurite outgrowth and synaptogenesis, supporting a role for the receptor in these processes. Collectively, these data suggest that Met signaling may be necessary for the appropriate wiring of forebrain circuits, with particular relevance to the social and emotional dimensions of behavior. J. Comp. Neurol. 513:511–531, 2009.

Allelic Specificity of Ube3a Expression in the Mouse Brain during Postnatal Development

Genetic alterations of the maternal UBE3A allele result in Angelman syndrome (AS), a neurodevelopmental disorder characterized by severe developmental delay, lack of speech, and difficulty with movement and balance. The combined effects of maternal UBE3A mutation and cell type–specific epigenetic silencing of paternal UBE3A are hypothesized to result in a complete loss of functional UBE3A protein in neurons. However, the allelic specificity of UBE3A expression in neurons and other cell types in the brain has yet to be characterized throughout development, including the early postnatal period when AS phenotypes emerge. Here we define maternal and paternal allele‐specific Ube3a protein expression throughout postnatal brain development in the mouse, a species that exhibits orthologous epigenetic silencing of paternal Ube3a in neurons and AS‐like behavioral phenotypes subsequent to maternal Ube3a deletion. We find that neurons downregulate paternal Ube3a protein expression as they mature and, with the exception of neurons born from postnatal stem cell niches, do not express detectable paternal Ube3a beyond the first postnatal week. By contrast, neurons express maternal Ube3a throughout postnatal development, during which time localization of the protein becomes increasingly nuclear. Unlike neurons, astrocytes and oligodendrotyes biallelically express Ube3a. Notably, mature oligodendrocytes emerge as the predominant Ube3a‐expressing glial cell type in the cortex and white matter tracts during postnatal development. These findings demonstrate the spatiotemporal characteristics of allele‐specific Ube3a expression in key brain cell types, thereby improving our understanding of the developmental parameters of paternal Ube3a silencing and the cellular basis of AS. J. Comp. Neurol. 522:1874–1896, 2014.

EVIDENCE OF CELL-NONAUTONOMOUS CHANGES IN DENDRITE AND DENDRITIC SPINE MORPHOLOGY IN THE MET-SIGNALING DEFICIENT MOUSE FOREBRAIN

Human genetic findings and murine neuroanatomical expression mapping have intersected to implicate Met receptor tyrosine kinase signaling in the development of forebrain circuits controlling social and emotional behaviors that are atypical in autism‐spectrum disorders (ASD). To clarify roles for Met signaling during forebrain circuit development in vivo, we generated mutant mice (Emx1Cre/Metfx/fx) with an Emx1‐Cre‐driven deletion of signaling‐competent Met in dorsal pallially derived forebrain neurons. Morphometric analyses of Lucifer yellow‐injected pyramidal neurons in postnatal day 40 anterior cingulate cortex (ACC) revealed no statistically significant changes in total dendritic length but a selective reduction in apical arbor length distal to the soma in Emx1Cre/Metfx/fx neurons relative to wild type, consistent with a decrease in the total tissue volume sampled by individual arbors in the cortex. The effects on dendritic structure appear to be circuit‐selective, insofar as basal arbor length was increased in Emx1Cre/Metfx/fx layer 2/3 neurons. Spine number was not altered on the Emx1Cre/Metfx/fx pyramidal cell populations studied, but spine head volume was significantly increased (∼20%). Cell‐nonautonomous, circuit‐level influences of Met signaling on dendritic development were confirmed by studies of medium spiny neurons (MSN), which do not express Met but receive Met‐expressing corticostriatal afferents during development. Emx1Cre/Metfx/fx MSN exhibited robust increases in total arbor length (∼20%). As in the neocortex, average spine head volume was also increased (∼12%). These data demonstrate that a developmental loss of presynaptic Met receptor signaling can affect postsynaptic morphogenesis and suggest a mechanism whereby attenuated Met signaling could disrupt both local and long‐range connectivity within circuits relevant to ASD. J. Comp. Neurol. 518:4463–4478, 2010.

A new synaptic player leading to autism risk: Met receptor tyrosine kinase

The validity for assigning disorder risk to an autism spectrum disorder (ASD) candidate gene comes from convergent genetic, clinical, and developmental neurobiology data. Here, we review these lines of evidence from multiple human genetic studies, and non-human primate and mouse experiments that support the conclusion that the MET receptor tyrosine kinase (RTK) functions to influence synapse development in circuits relevant to certain core behavioral domains of ASD. There is association of both common functional alleles and rare copy number variants that impact levels of MET expression in the human cortex. The timing of Met expression is linked to axon terminal outgrowth and synaptogenesis in the developing rodent and primate forebrain, and both in vitro and in vivo studies implicate this RTK in dendritic branching, spine maturation, and excitatory connectivity in the neocortex. This impact can occur in a cell-nonautonomous fashion, emphasizing the unique role that Met plays in specific circuits relevant to ASD.

Conserved Subcortical and Divergent Cortical Expression of Proteins Encoded by Orthologs of the Autism Risk Gene MET

Met receptor tyrosine kinase signaling regulates the growth and development of axons and may contribute to the wiring of cortical and limbic circuits in the rodent forebrain. Whether the orthologous MET receptor functions similarly in the developing primate forebrain is not known but is of considerable interest considering the association of variant MET alleles with social and communication phenotypes in autism. To begin addressing this question, we compared Met/MET protein expression in the developing mouse and rhesus macaque forebrain. There was a strong temporal conservation of expression during the time of rapid axon development and the onset of robust synapse formation. Expression patterns of Met/MET in limbic-related structures were almost identical between species. In marked contrast, there was highly divergent expression in the neocortex. In mouse, Met was broadly distributed throughout neocortex. In the macaque, robust MET expression was largely restricted to the posterior cingulate, inferior temporal, posterior parietal, and visual cortices, including face processing regions. The pattern is consistent with the importance of vision in the social repertoire of the primate. Collectively, these data suggest a conserved developmental function of the MET receptor in wiring together limbic and neocortical circuits that facilitate species-appropriate responses, including social behavior.

Loss of UBE3A from TH-expressing neurons suppresses GABA co-release and enhances VTA-NAc optical self-stimulation

Motivated reward-seeking behaviours are governed by dopaminergic ventral tegmental area projections to the nucleus accumbens. In addition to dopamine, these mesoaccumbal terminals co-release other neurotransmitters including glutamate and GABA, whose roles in regulating motivated behaviours are currently being investigated. Here we demonstrate that loss of the E3-ubiquitin ligase, UBE3A, from tyrosine hydroxylase-expressing neurons impairs mesoaccumbal, non-canonical GABA co-release and enhances reward-seeking behaviour measured by optical self-stimulation.

Dissociation of locomotor and cerebellar deficits in a murine Angelman syndrome model

Angelman syndrome (AS) is a severe neurological disorder that is associated with prominent movement and balance impairments that are widely considered to be due to defects of cerebellar origin. Here, using the cerebellar-specific vestibulo-ocular reflex (VOR) paradigm, we determined that cerebellar function is only mildly impaired in the Ube3am-/p+ mouse model of AS. VOR phase-reversal learning was singularly impaired in these animals and correlated with reduced tonic inhibition between Golgi cells and granule cells. Purkinje cell physiology, in contrast, was normal in AS mice as shown by synaptic plasticity and spontaneous firing properties that resembled those of controls. Accordingly, neither VOR phase-reversal learning nor locomotion was impaired following selective deletion of Ube3a in Purkinje cells. However, genetic normalization of αCaMKII inhibitory phosphorylation fully rescued locomotor deficits despite failing to improve cerebellar learning in AS mice, suggesting extracerebellar circuit involvement in locomotor learning. We confirmed this hypothesis through cerebellum-specific reinstatement of Ube3a, which ameliorated cerebellar learning deficits but did not rescue locomotor deficits. This double dissociation of locomotion and cerebellar phenotypes strongly suggests that the locomotor deficits of AS mice do not arise from impaired cerebellar cortex function. Our results provide important insights into the etiology of the motor deficits associated with AS.

Subcellular organization of UBE3A in neurons

Ubiquitination regulates a broad array of cellular processes, and defective ubiquitination is implicated in several neurological disorders. Loss of the E3 ubiquitin–protein ligase UBE3A causes Angelman syndrome. Despite its clinical importance, the normal role of UBE3A in neurons is still unclear. As a step toward deciphering its possible functions, we performed high‐resolution light and electron microscopic immunocytochemistry. We report a broad distribution of UBE3A in neurons, highlighted by concentrations in axon terminals and euchromatin‐rich nuclear domains. Our findings suggest that UBE3A may act locally to regulate individual synapses while also mediating global, neuronwide influences through the regulation of gene transcription. J. Comp. Neurol. 525:233–251, 2017.

Decreased Axon Caliber Underlies Loss of Fiber Tract Integrity, Disproportional Reductions in White Matter Volume, and Microcephaly in Angelman Syndrome Model Mice

Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of function of the maternally inherited UBE3A allele. It is currently unclear how the consequences of this genetic insult unfold to impair neurodevelopment. We reasoned that by elucidating the basis of microcephaly in AS, a highly penetrant syndromic feature with early postnatal onset, we would gain new insights into the mechanisms by which maternal UBE3A loss derails neurotypical brain growth and function. Detailed anatomical analysis of both male and female maternal Ube3a-null mice reveals that microcephaly in the AS mouse model is primarily driven by deficits in the growth of white matter tracts, which by adulthood are characterized by densely packed axons of disproportionately small caliber. Our results implicate impaired axon growth in the pathogenesis of AS and identify noninvasive structural neuroimaging as a potentially valuable tool for gauging therapeutic efficacy in the disorder. SIGNIFICANCE STATEMENT People who maternally inherit a deletion or nonfunctional copy of the UBE3A gene develop Angelman syndrome (AS), a severe neurodevelopmental disorder. To better understand how loss of maternal UBE3A function derails brain development, we analyzed brain structure in a maternal Ube3a knock-out mouse model of AS. We report that the volume of white matter (WM) is disproportionately reduced in AS mice, indicating that deficits in WM development are a major factor underlying impaired brain growth and microcephaly in the disorder. Notably, we find that axons within the WM pathways of AS model mice are abnormally small in caliber. This defect is associated with slowed nerve conduction, which could contribute to behavioral deficits in AS, including motor dysfunction.