Ube3a Role in Synaptic Plasticity and Neurodevelopmental Disorders.The Lessons from Angelman Syndrome.

Angelman Syndrome (AS) is a severe neurodevelopmental disorder that affects 1:12000 newborns. It is characterized by mental retardation, delayed major motor and cognitive milestones, seizures, absence of speech and excessive laughter. The majority of AS cases arise from deletions or mutations of UBE3A gene located on the chromosome 15q11-13. UBE3A codes for E3-ubiquitin ligase that target specific proteins for degradation. To date, a wide variety of Ube3a substrates has been identified. The accumulation of Ube3a-dependent proteins and their effect on the multitude of signal transduction pathways are` considered the main cause of the AS pathology. While the majority of research has been directed towards target identifications, the overall role of Ube3a in activity-dependent synaptic plasticity has been greatly overlooked. The present work is designed to fill some of these knowledge gaps. Chapter 2 is focused on the activity-dependent aspect of Ube3a expression following neuronal stimulation in vivo and in vitro. We examined total Ube3a expression followed by KCl depolarization in neuronal primary culture. By utilizing a subcellular fractionation technique, we were able to determine which cellular pools are responsive to the depolarization. Next, a fear conditioning paradigm (FC) was used to activate neurons in the paternal Ube3a-YFP reporter mouse brain. This mouse model allowed us to resolve spatial and temporal alterations of the maternal and the paternal Ube3a in hippocampus and cortex followed by FC. In accordance to KCl depolarization results, we observed alterations in Ube3a protein but at later time points. Furthermore, we investigated if the absence of activity-dependent Ube3a changes has any effect on learning and memory kinase activation. We utilized KCl and FC to determine synaptic activity-induced ERK 1/2 phosphorylation in acute hippocampal slices and in CA1 area of hippocampus of wild type (Ube3a m+/p+) and Ube3a deficient mice (Ube3a m-/p+). We demonstrated that Ube3a loss leads to impaired activity-dependent ERK 1/2 phosphorylation. It has been established that Ube3a m-/p+ mice have a profound deficit in LTP, implying the importance of this ligase in excitatory synaptic transmission. The abnormal LTP could be partially explained by an aberrant CaMKII function, decreased activity-dependent ERK 1/2 phosphorylation and reduced phosphatase activity. These proteins have also been implicated in another form of synaptic plasticity such as long-term depression (LTD). Chapter 3, we investigated the contribution of Ube3a to NMDAR - dependent and - independent LTD. Our data showed that Ube3a m-/p+ P21-30 animals exhibit the impairments in both forms of LTD. Next, we focused on elucidating molecular mechanism underlying the reduced mGluR1/5-LTD. We discovered that mGluR1/5 kinase activation such as ERK, mTOR and p38 is not affected by Ube3a loss. In concordance with previous work, we detected increased Arc expression together with abnormal AMPAR distribution in the Ube3a m-/p+ hippocampus. Surprisingly, the mGluR1/5 induced GluR2 trafficking was normal. Our findings infer that elevated Arc levels together with the increased internalization of AMPAR may result in compromised basal state of the synapses leading to a more depression-like state in Ube3a m-/p+ mice. Evidence points that loss of Ube3a produces alterations in a variety of activity-dependent signal transduction cascades that may ultimately result in impaired synaptic plasticity and cognition. Similar to AS, abnormal molecular and behavioral phenotypes have already been observed in other mouse models of human mental retardation such as Fragile X Mental Retardation Syndrome (FXS). Chapter 4 is set to explore if any correlation can be found in between these neurodevelopmental disorders. Analysis of crude synaptoneurosomes of adult Fmr1 KO mice revealed a significant reduction in Ube3a protein. Additionally, a blunted translation of Ube3a in response to mGluR1/5 stimulation was observed. However, we didn\u27t find any evidence of direct interaction between Ube3a mRNA and Fragile X Mental Retardation Protein (FMRP). To examine if some of the pathology seen in Fmr1 KO mice is due to Ube3a downregulation, we performed a rescue experiment by increasing overall levels of Ube3a in hippocampus of FRMP deficient mice. An exhaustive battery of behavioral testing indicated that alterations of Ube3a expression impacted only associative fear conditioning. In summary, the present work has attempted to answer some of the fundamental questions about Ube3a and its role in synaptic plasticity. We have demonstrated that Ube3a expression is modulated by synaptic activation and its activity-dependent alterations are essential for normal brain functioning. Additionally, our data suggest that Ube3a is not only significant for the synaptic excitation but also crucial for the synaptic depression. Finally, our findings indicate that the alteration of Ube3a expression may contribute to the cognitive phenotypes in other neurodevelopmental disorders such as FXS suggesting an advantage of exploring Ube3a function outside the AS research