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Novel target for Angelman Syndrome and Autism spectrum disorders

Background: Angleman Syndrome (AS) is a neuro-genetic disorder characterized by intellectual and developmental delay as well as sleep disturbance, seizure and trembling movements. This debilitating neurological disorder is caused by mutation of the E3 ubiquitin ligase (Ube3A), a gene whose mutation has also recently been associated with autism spectrum disorders (ASD). However, the function of Ube3A in mediating cognitive impairment in individuals with AS and ASDs, as well as its substrates, have been unknown.

Invention: The Greenberg laboratory first demonstrated that neural activity induces Ube3A transcription, and that a decrease in Ube3A expression decreases the plasma membrane expression of, and synaptic transmission through AMPA glutamate receptors (AMPARs). To better understand the role of Ube3A in AS and ASD, the Greenberg lab identified key neural substrates of Ube3A, Arc and Ephexin5, and the mechanisms for their regulation of synaptic transmission. Their findings suggest mechanisms by which Ube3A contributes to cognitive dysfunction in AS and ASD.

• Arc is a substrate of Ube3A. The Greenberg lab searched mammalian genomes for proteins that contain a Ube3A binding domain. Their search identified Arc, a protein known to rapidly increase upon glutamate release at excitatory synapses, which regulates the trafficking and expression of AMPARs at synapses. Arc-mediated endocytosis of AMPARs is known to dampen neuronal excitability, and in turn, limit the level of neuronal excitation. The role of Ube3A may therefore be to bind to and regulate Arc-mediated endocytosis to maintain proper synaptic activation.
• Ube3A controls synaptic function by ubiquitinating and degrading Arc. They next showed that Ube3A controls the level of Arc protein expression by ubiquitinating and thus targeting Arc for degradation. In this way, Ube3A reduces the Arc-mediated internalization of AMPARs.

• Ephexin5 negatively regulates excitatory synapse development. Recent research reveals an important role for Eph receptor tyrosine kinases in the early cell-cell contact phase that is critical for excitatory synapse formation. EphBs are highly expressed in neurons, and are positive regulators of excitatory synapse development by triggering the recruitment of AMPA and NMDA subtypes of excitatory glutamate receptors.
The inventors identified a RhoA guanine nucleotide exchange factor, Ephexin5, which interacts with EphB2 as determined by co-immunoprecipitation, and co-localization using immunofluorescence microscopy. In addition, they showed that Ephexin5 negatively regulates excitatory synapse development by knocking down the levels of Ephexin5 protein expression in cultured neurons, which resulted in a significant increase in the number of excitatory synapses. The lab also demonstrated that Ephexin5 functions directly to restrict the synapse-promoting effects of EphB2, suggesting that in order for EphB2 to positively regulate synapse development, it is necessary to inactivate and/or degrade Ephexin5.
• Ephexin5 is a substrate of Ube3A. The inventors showed that Ephexin5 co-immunoprecipitates with Ube3A but not with other E3 ligases. In addition, they identified a Ube3A binding domain in Ephexin5.
• Ube3A ubiquitinates and degrades Ephexin5. The lab demonstrated that EphB triggers the phosphorylation of specific tyrosine residues on Ephexin5. Ubiquitination and degradation of Ephexin5 is then mediated by Ube3A, which promotes EphB-dependent excitatory synapse development. Their results suggest that phosphorylation of Ephexin5 by EphB is required for the Ube3A-mediatied degradation.

• Ube3A’s role in cognitive dysfunction. The lab used a mouse model of Angelman syndrome, which is deficient in Ube3A, to further elucidate the role of Ube3A in cognitive dysfunction. This mouse was shown to have elevated levels of Arc in neurons, resulting in excessive internalization of AMPARs at synapses and impaired synaptic transmission. The disregulation of AMPAR expression may contribute to the cognitive dysfunction in AS and possibly other ASDs.
This mouse model also has decreased levels of ubiquitinated Ephexin5 and increased levels of Ephexin5 protein expression. These findings suggest that aberrant EphB/Ephexin5 signaling, leading to a disruption in EphB-dependent excitatory synapse development and function, contributes to the abnormal cognitive function that occurs in AS and possibly other ASDs.

Intellectual Property Status: Patent(s) Pending

Applications

Angelman syndrome (AS) and Autistic spectrum disorders (ASD): Although it has been known for more than a decade that mutation of Ube3A results in AS, remarkably little is understood about the role of Ube3A in the cognitive impairment underlying AS. This lack of insight has hampered the development of therapeutic strategies for treating AS, and as a result there are currently no effective treatments for this disorder. This research pinpoints potential therapeutic targets for the development of therapies to treat diseases associated with mutations in Ube3A including AS and ASD.

Arc: The Greenberg lab demonstrated that disruption of Ube3A activity leads to an increase of Arc and decrease in AMPAR expression at synapses. Drugs that promote AMPAR expression at synapses, such as metabotropic glutamate receptor subtype 5 (mGluR5) antagonists or compounds that inhibit the expression or function or Arc, may reverse symptoms associated with AS and ASD.
Fragile X is a human disorder in which a similar decrease in AMPAR expression at synapses has been demonstrated. This decrease has further been shown to be a result of excessive mGluR5 signaling, resulting in increased Arc translation and excessive AMPAR internalization. Selective mGluR5 antagonists are now entering clinical trials for the treatment of Fragile X, indicating that this type of therapeutic strategy has potential

Ephexin5: Disruption of Ube3A also results in an increase of Ephexin5, leading to aberrant EphB/Ephexin5 signaling, and a disruption of EphB-dependent excitatory synapse development. Compounds that inhibit Ephexin5 activity and stimulate excitatory synapse formation, may reverse symptoms associated with AS and ASD.

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