Synaptic function and integrity in the nervous system requires the expression of synaptic adhesion molecules (SAMs) linking pre- and post-synaptic sites. Studies indicate that SAMs participate in the formation, maturation, function and plasticity of synaptic connections, and thus are essential for trans-cellular signaling. Alterations in SAMs lead to susceptibility to neurological diseases including the autism spectrum disorders, schizophrenia, and addiction. Neurexins compose a family of highly polymorphic type I transmembrane proteins that are expressed on the pre-synaptic membrane at excitatory glutamatergic and inhibitory GABAergic synapses. Beta-neurexins have a single folding domain in the extracellular region, while alpha-neurexins have a larger extracellular region containing nine independently folding domains and multiple protein interaction sites. They both function as adhesion molecules through a trans- synaptic complex with post-synaptic neuroligins. Synaptogenesis and synapse function requires the precise assembly of pre- and post-synaptic protein complexes. The work described herein uses structural and biophysical techniques to discern the molecular properties of the neurexin and neuroligin proteins that mediate their complex formation. The first aim included solving the X- ray crystal structure of the beta-neurexin:neuroligin complex, which showed a stable neuroligin dimer and two monomeric beta-neurexin molecules bound on either side of the dimer. This structure revealed the molecular adhesion properties of the complex, including a Ca²⁺-coordination site. Alpha- and beta-neurexins have the same binding domain for neuroligins, yet they are likely to act as functionally distinct molecules in the synapse. To consider their distinctive adhesion properties, as well as how the multi-domain alpha-neurexins assemble in the limited space of a synapse, the second and third aims were directed at solving the 3D structure of alpha-neurexin. First, small angle X-ray scattering and single-particle negative-stain electron microscopy provided information on the overall domain organization and flexibility of the protein. This work led to the high-resolution crystal structure of a major portion of the alpha-neurexin extracellular region. The crystal structure reveals molecular details that suggest a multi-functional mechanism of the alpha-neurexin extracellular region in the synapse. Overall, this work contributes to the understanding of how synapses assemble through a complex network of protein-protein interactions and provides structural templates for the development of molecular tools to study function and potentially therapeutic applications

Angelman Syndrome (AS) is a severe neurodevelopmental disorder due to impaired expression of UBE3A in neurons. There are several genetic mechanisms that impair UBE3A expression, but they differ in how neighboring genes on chromosome 15 at 15q11-q13 are affected. There is evidence that different genetic subtypes present with different clinical severity, but a systematic quantitative investigation is lacking. Here we analyze natural history data on a large sample of individuals with AS (n = 250, 848 assessments), including clinical scales that quantify development of motor, cognitive, and language skills (Bayley Scales of Infant Development, Third Edition; Preschool Language Scale, Fourth Edition), adaptive behavior (Vineland Adaptive Behavioral Scales, Second Edition), and AS-specific symptoms (AS Clinical Severity Scale). We found that clinical severity, as captured by these scales, differs between genetic subtypes: individuals with UBE3A pathogenic variants and imprinting defects (IPD) are less affected than individuals with uniparental paternal disomy (UPD); of those with UBE3A pathogenic variants, individuals with truncating mutations are more impaired than those with missense mutations. Individuals with a deletion that encompasses UBE3A and other genes are most impaired, but in contrast to previous work, we found little evidence for an influence of deletion length (class I vs. II) on severity of manifestations. The results of this systematic analysis highlight the relevance of genomic regions beyond UBE3A as contributing factors in the AS phenotype, and provide important information for the development of new therapies for AS. More generally, this work exemplifies how increasing genetic irregularities are reflected in clinical severity.

Angelman syndrome (AS) is a complex, heterogeneous, and life-long neurodevelopmental disorder. Despite the considerable impact on individuals and caregivers, no disease-modifying treatments are available. To support holistic clinical management and the development of AS-specific outcome measures for clinical studies, we conducted primary and secondary research identifying the impact of symptoms on individuals with AS and their unmet need. This qualitative research adopted a rigorous step-wise approach, aggregating information from published literature, then evaluating it via disease concept elicitation interviews with clinical experts and caregivers. We found that the AS-defining concepts most relevant for treatment included: impaired expressive communication, seizures, maladaptive behavior, cognitive impairment, motor function difficulties, sleep disturbance, and limited self-care abilities. We highlight the relevance of age in experiencing these key AS concepts, and the difference between the perceptions of clinicians and caregivers towards the syndrome. Finally, we outline the impact of AS on individuals, caregivers, and families.

Rett syndrome (RTT) is a pervasive developmental disorder caused by mutations in MECP2. Complete loss of MECP2 function in males causes congenital encephalopathy, neurodevelopmental arrest, and early lethality. Induced pluripotent stem cell (iPSC) lines from male patients harboring mutations in MECP2, along with control lines from their unaffected fathers, give us an opportunity to identify some of the earliest cellular and molecular changes associated with MECP2 loss-of-function (LOF). We differentiated iPSC-derived neural progenitor cells (NPCs) using retinoic acid (RA) and found that astrocyte differentiation is perturbed in iPSC lines derived from two different patients. Using highly stringent quantitative proteomic analyses, we found that LIN28, a gene important for cell fate regulation and developmental timing, is upregulated in mutant NPCs compared to WT controls. Overexpression of LIN28 protein in control NPCs suppressed astrocyte differentiation and reduced neuronal synapse density, whereas downregulation of LIN28 expression in mutant NPCs partially rescued this synaptic deficiency. These results indicate that the pathophysiology of RTT may be caused in part by misregulation of developmental timing in neural progenitors, and the subsequent consequences of this disruption on neuronal and glial differentiation.

BACKGROUND:Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by either disruptions of the gene UBE3A or deletion of chromosome 15 at 15q11-q13, which encompasses UBE3A and several other genes, including GABRB3, GABRA5, GABRG3, encoding gamma-aminobutyric acid type A receptor subunits (β3, α5, γ3). Individuals with deletions are generally more impaired than those with other genotypes, but the underlying pathophysiology remains largely unknown. Here, we used electroencephalography (EEG) to test the hypothesis that genes other than UBE3A located on 15q11-q13 cause differences in pathophysiology between AS genotypes. METHODS:We compared spectral power of clinical EEG recordings from children (1-18 years of age) with a deletion genotype (n = 37) or a nondeletion genotype (n = 21) and typically developing children without Angelman syndrome (n = 48). RESULTS:We found elevated theta power (peak frequency: 5.3 Hz) and diminished beta power (peak frequency: 23 Hz) in the deletion genotype compared with the nondeletion genotype as well as excess broadband EEG power (1-32 Hz) peaking in the delta frequency range (peak frequency: 2.8 Hz), shared by both genotypes but stronger for the deletion genotype at younger ages. CONCLUSIONS:Our results provide strong evidence for the contribution of non-UBE3A neuronal pathophysiology in deletion AS and suggest that hemizygosity of the GABRB3-GABRA5-GABRG3 gene cluster causes abnormal theta and beta EEG oscillations that may underlie the more severe clinical phenotype. Our work improves the understanding of AS pathophysiology and has direct implications for the development of AS treatments and biomarkers.