A Behavioral and Molecular Approach for Understanding Angelman Syndrome

Autism Spectrum Disorder (ASD) is a set of human developmental disorders that affects ~1 in 68 children. The clinical features of ASD include deficits in social behavior and frequent co-morbidity of motor, emotional and sensory impairment. Currently, there are no effective treatments. A major obstacle for treating ASD is the limited knowledge of the neuronal circuits that drive these complex behaviors. Several monogenic, or single-gene, disorders that possess similar features to ASD have been identified, implicating a role for molecular pathways in the development of these behavioral circuits. This dissertation focuses on Angelman Syndrome (AS), a neurodevelopmental disorder characterized by social communication deficits, movement disorder and hyper-excitable behavioral traits. The phenotype of AS arises from mutation of the UBE3A gene, encoding an E3 ubiquitin ligase. The overarching goal of this study is to understand how deregulation of UBE3A-dependent pathways contribute to the behavioral phenotype of AS. Neuronal substrates of UBE3A have been identified and their expression has been shown to be up-regulated in AS neurons. I now test the hypothesis that this deregulation contributes to specific pathology of AS. First, I described clinically relevant behavioral phenotypes in an AS mouse model. Next, I genetically reduced the expression level of two UBE3A substrates, ARC (Activity-Regulated Cytoskeleton-Associated Protein) and EPHEXIN5 (Rho Guanine Nucleotide Exchange Factor 15) in the AS mouse and assayed for reversal of behavioral abnormalities. I find that the AS mouse model has impaired communication and motor behavior during early postnatal development, enhanced seizure-like activity and an abnormal cortical electroencephalogram (EEG). Reducing the levels of ARC reversed the enhanced seizure-like activity and EEG, but not the communication or motor deficits. The specific rescue of seizure-like activity by reducing ARC, but not EPHEXIN5, reveals a role for molecular diversity in the development of behavioral circuits. Further, these findings suggest that therapeutic interventions that reduce the level of ARC expression have the potential to reverse the seizures associated with AS. Lastly, the identification of aberrant behaviors in AS mice provides clues regarding the neural circuit defects that occur in AS and ultimately allow new approaches for treating this disorder, and broader ASDs.Medical Science

Altered Dopamine Signaling in Naturally Occurring Maternal Neglect

Child neglect is the most common form of child maltreatment, yet the biological basis of maternal neglect is poorly understood and a rodent model is lacking.The current study characterizes a population of mice (MaD1) which naturally exhibit maternal neglect (little or no care of offspring) at an average rate of 17% per generation. We identified a set of risk factors that can predict future neglect of offspring, including decreased self-grooming and elevated activity. At the time of neglect, neglectful mothers swam significantly more in a forced swim test relative to nurturing mothers. Cross-fostered offspring raised by neglectful mothers in turn exhibit increased expression of risk factors for maternal neglect and decreased maternal care as adults, suggestive of possible epigenetic contributions to neglect. Unexpectedly, offspring from neglectful mothers elicited maternal neglect from cross-fostered nurturing mothers, suggesting that factors regulating neglect are not solely within the mother. To identify a neurological pathway underlying maternal neglect, we examined brain activity in neglectful and nurturing mice. c-Fos expression was significantly elevated in neglectful relative to nurturing mothers in the CNS, particularly within dopamine associated areas, such as the zona incerta (ZI), ventral tegmental area (VTA), and nucleus accumbens. Phosphorylated tyrosine hydroxylase (a marker for dopamine production) was significantly elevated in ZI and higher in VTA (although not significantly) in neglectful mice. Tyrosine hydroxylase levels were unaltered, suggesting a dysregulation of dopamine activity rather than cell number. Phosphorylation of DARPP-32, a marker for dopamine D1-like receptor activation, was elevated within nucleus accumbens and caudate-putamen in neglectful versus nurturing dams.These findings suggest that atypical dopamine activity within the maternal brain, especially within regions involved in reward, is involved in naturally occurring neglect and that MaD1 mice are a useful model for understanding the basis of naturally occurring neglect

Molecular mimicry in multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a severe, post-infectious sequela of SARS-CoV-2 infection1,2, yet the pathophysiological mechanism connecting the infection to the broad inflammatory syndrome remains unknown. Here we leveraged a large set of samples from patients with MIS-C to identify a distinct set of host proteins targeted by patient autoantibodies including a particular autoreactive epitope within SNX8, a protein involved in regulating an antiviral pathway associated with MIS-C pathogenesis. In parallel, we also probed antibody responses from patients with MIS-C to the complete SARS-CoV-2 proteome and found enriched reactivity against a distinct domain of the SARS-CoV-2 nucleocapsid protein. The immunogenic regions of the viral nucleocapsid and host SNX8 proteins bear remarkable sequence similarity. Consequently, we found that many children with anti-SNX8 autoantibodies also have cross-reactive T cells engaging both the SNX8 and the SARS-CoV-2 nucleocapsid protein epitopes. Together, these findings suggest that patients with MIS-C develop a characteristic immune response to the SARS-CoV-2 nucleocapsid protein that is associated with cross-reactivity to the self-protein SNX8, demonstrating a mechanistic link between the infection and the inflammatory syndrome, with implications for better understanding a range of post-infectious autoinflammatory diseases

Phage display demonstrates durable differences in serological profile by route of inoculation in primary infections of non-human primates with Dengue Virus 1.

Natural dengue virus (DENV) infections occur by mosquito bite but how the inoculation route affects the humoral immune response is unknown. We serologically profiled 20 non-human primates (NHP) from a prior study of DENV1 infection where animals were inoculated by mosquito (N = 10) or subcutaneous injection (N = 10). Using a comprehensive, densely tiled and highly redundant pan-flavivirus programmable phage library containing 91,562 overlapping 62 amino acid peptides, we produced a high-resolution map of linear peptide sequences enriched during DENV seroconversion. Profiles in mosquito-inoculated and subcutaneously-inoculated animals were similar up to 90 days after primary infection, but diverged at 1 year with differences in sero-reactivity in the Envelope (E; residues 215-406; p < 0.08), and Nonstructural-3 (NS3; residues 549-615; p < 0.05) proteins in mosquito-inoculated versus subcutaneously-inoculated animals. Within the E protein, residues 339-384 in domain III accounted for > 99% of the observed sero-reactivity difference. Antibody breadth did not vary by mode of inoculation. The differential reactivity to E domain III seen by phage display validated orthogonally by ELISA, but did not correlate with late neutralization titers. Serological profiling of humoral immune responses to DENV infection in NHP by programmable phage display demonstrated durable differences in sero-reactivity by route of inoculation

Mean (±SE) number of c-Fos positive cells in additional brain regions in neglectful and nurturing mice.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001974#pone-0001974-g006" target="_blank">Fig. 6</a> for c-Fos in dopamine releasing and responding regions.</p><p>Abbreviations: LSV = lateral septum ventral; CG = cingulate cortex; MeA = medial amygdala; CeA = central amygdala; LH = lateral hypothalamus; Pir = piriform cortex; PVN = paraventricular nucleus; cPAG = caudal periaqueductal gray; cPAG1 = more caudal aspect of cPAG; MPOM = medial preoptic nucleus; MPA = medial preoptic area; AHA = anterior hypothalamic area; SCN = suprachiasmatic nucleus. #- data non-normal, analyzed with ANOVA on Ranks.</p

Altered pDARPP-32 expression with maternal neglect.

<p>Previously neglectful dams (N = 5) have significantly darker optical density of pDARPP-32 expression in Ac and CP when compared to previously nurturing dams (N = 6). Ac = nucleus accumbens, LS = lateral septum, CP = caudate-putamen, BST = bed nucleus of stria terminalis, dorsal, CeA = central amygdala. Bars represent means±SE. *  = p<0.05.</p