Angelman Syndrome (AS) is a severe neurodevelopmental disorder affecting approximately 1:20.000 newborn children. AS mainly affects the central nervous system and causes severe physical and learning disabilities. Children appear entirely normal at birth, but after the first year of life developmental milestones are not met and development entirely stalls at a developmental age of 2 years, regardless of the actual age of the patient. Symptoms may vary between patients, but most commonly include lack of speech, severe intellectual disability, motor deficits, behavioral abnormalities, severely disrupted sleep and seizures. AS patients have a normal life expectancy, but need lifelong daily care.
AS is caused by loss of a functional UBE3A protein. The preclinical research at the ENCORE expertise center focusses on understanding UBE3A function and disfunction, with the ultimate goal to develop potential treatments to ameliorate AS symptoms.
Identifying UBE3A targets.
UBE3A (originally identified as E6-associated protein, E6-AP) is an enzyme with ubiquitin E3 ligase activity. That means it adds ubiquitin molecules to other proteins (called ‘target’ protein). Typically, adding a ubiquitin (Ub) molecule to a target protein will change the activity of the target protein or result in the breakdown of the target protein. An important line of research is to identify the target proteins that are modified by UBE3A. These targets will provide us with important information about the precise role of UBE3A in neuronal function.
Understanding the effect of UBE3A mutations.
In a subset of patients we find mutations in the UBE3A gene that look quite subtle in which only a single amino acid is replaced by another amino acid (called ‘missense mutation’). This is comparable to a spelling mistake in a word. What is the effect of such a mistake? Sometimes the effect is very severe, and the patients shows a classical Angelman Syndrome phenotype. In other cases, the patient does not look like a typical AS patient, and we are not sure if UBE3A is actually malfunctioning or if a different gene is mutated (See for instance Geerts-Haages, Molecular Genetics and Genome Medicine, 2020). We have now developed assays to measure the effect of missense mutations on the UBE3A protein, by developing a UBE3A ubiquitination assay. This will help us in diagnosing new patients, and it will help us to understand how UBE3A functions.
The role of nuclear UBE3A.
We have recently shown that the mouse makes two slightly different UBE3A proteins (called isoform proteins). These proteins are slightly different in size. The shorter UBE3A protein is specifically localized in the nucleus, the location where the DNA is stored and read. The larger UBE3A protein is present everywhere outside the nucleus (called cytosol). We have recently discovered how this transport to the nucleus is achieved (Avagliano-Trezza, Nature Neuroscience 2019). We further found that in human and mice, most of the UBE3A is located in the nucleus (Zampeta, Human Molecular Genetics 2020), and we have shown that the nuclear UBE3A protein is most important for brain function. A major research line of the lab is now to study the role of UBE3A in the nucleus.
Understanding the role of UBE3A in the brain.
We also study the role of UBE3A in brain function and development. Given that the living human brain is an organ that cannot easily be studied, we use a mouse model for AS that has several symptoms as observed in AS patients (epilepsy, motor deficits, increased anxiety, repetitive behavior). Like the patients, this mouse is lacking a functional UBE3A gene.
An additional advantage of our mouse model system lays in a genetic trick, such that we can switch the UBE3A gene on or off at any given time. We can also switch the gene on or off in specific parts of the brain (Silva-Santos, J. Clinical Investigation, 2015; Sonzogni, Molecular Autism 2019, 2020). This enables us to study its role in brain development, and also determine which brain area is mostly affected. Using electrophysiology, the produced electrical signals in the brain can be assessed and compared between AS and control mice and can be correlated to the behavior of these mice. We are currently focusing on the role of UBE3A in brain development and on the role of UBE3A in the striatum, a specific part of the brain that is possibly responsible for motor and behavioral phenotypes.
Towards a treatment for AS.
In addition to fundamental studies on UBE3A and its relation to AS, we are also investigating potential therapeutic approaches to alleviate the symptoms of AS patients. To that purpose, we have developed a standardized screening protocol which we use to assess the effect of drugs on AS mouse behavior (Sonzogni, Molecular Autism, 2018). We are continuously testing novel drugs that can potentially correct neuronal function. Besides conventional (small molecule) therapies, we are also employing genetic strategies to restore UBE3A function. This includes the use of antisense oligonucleotides (ASOs) to activate the (paternal) UBE3A gene. ASOs are small pieces of DNA/RNA that can bind RNA molecules. In the case of Angelman Syndrome, these ASOs can restore the synthesis of UBE3A, by targeting the Ube3a-ATS RNA which is responsible of repressing the paternal UBE3A gene. Breakdown of the Ube3a-ATS RNA would restore UBE3A expression in AS patients.
Besides conventional (small molecule) therapies, we are also employing genetic strategies to restore UBE3A function. This includes the use of antisense oligonucleotides (ASOs) to activate the (paternal) UBE3A gene. ASOs are small pieces of DNA/RNA that can bind RNA molecules. In the case of Angelman Syndrome, these ASOs can restore the synthesis of UBE3A, by targeting the Ube3a-ATS RNA which is responsible of repressing the paternal UBE3A gene. Breakdown of the Ube3a-ATS RNA would restore UBE3A expression in AS patients.
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