Delayed loss of UBE3A reduces the expression of Angelman syndrome-associated phenotypes.

Background: Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by mutations affecting UBE3A gene expression. Previous studies in mice revealed distinct critical periods during neurodevelopment in which reactivation of Ube3a gene expression can prevent the onset of behavioral deficits. Whether UBE3A is required for brain function throughout life is unknown. Here, we address the importance of maintaining UBE3A expression after normal brain development. Findings: Using a conditional mouse, we deleted the Ube3a gene at three ages spanning brain maturation. We assessed the consequences of Ube3a gene deletion by testing the mice in behavioral tasks previously shown to produce robust phenotypes in AS model mice. Early embryonic deletion of Ube3a recapitulated all behavioral deficits of AS mice. In contrast, Ube3a gene deletion at 3 or 12 weeks of age did not have a significant effect on most behavioral tasks and did not increase seizure sensitivity. Conclusions: Taken together, these results emphasize that UBE3A critically impacts early brain development, but plays a more limited role in adulthood. Our findings provide important considerations for upcoming clinical trials in which UBE3A gene expression is reactivated and suggest that even transient UBE3A reinstatement during a critical window of early development is likely to prevent most adverse Angelman syndrome phenotypes. However, sustained UBE3A expression into adulthood is probably needed for optimal clinical benefit.

LRSAM1-mediated ubiquitylation is disrupted in axonal Charcot-Marie-Tooth disease 2P.

Charcot-Marie-Tooth (CMT) disease type 2 is a genetically heterogeneous group of inherited neuropathies characterized by motor and sensory deficits as a result of peripheral axonal degeneration. We recently reported a frameshift (FS) mutation in the Really Interesting New Gene finger (RING) domain of LRSAM1 (c.2121_2122dup, p.Leu708Argfs) that encodes an E3 ubiquitin ligase, as the cause of axonal-type CMT (CMT2P). However, the frequency of LRSAM1 mutations in CMT2 and the functional basis for their association with disease remains unknown. In this study, we evaluated LRSAM1 mutations in two large Dutch cohorts. In the first cohort (n = 107), we sequenced the full LRSAM1 coding exons in an unbiased fashion, and, in the second cohort (n = 468), we specifically sequenced the last, RING-encoding exon in individuals where other CMT-associated genes had been ruled out. We identified a novel LRSAM1 missense mutation (c.2120C > T, p.Pro707Leu) mapping to the RING domain. Based on our genetic analysis, the occurrence of pathogenic LRSAM1 mutations is estimated to be rare. Functional characterization of the FS, the identified missense mutation, as well as of another recently reported pathogenic missense mutation (c.2081G > A, p.Cys694Tyr), revealed that in vitro ubiquitylation activity was largely abrogated. We demonstrate that loss of the E2-E3 interaction that is an essential prerequisite for supporting ubiquitylation of target substrates, underlies this reduced ubiquitylation capacity. In contrast, LRSAM1 dimerization and interaction with the bona fide target TSG101 were not disrupted. In conclusion, our study provides further support for the role of LRSAM1 in CMT and identifies LRSAM1-mediated ubiquitylation as a common determinant of disease-associated LRSAM1 mutations.