The Ash Twins Blog

See previous posts on history, symptoms, neurophysiology, genetics, molecular biology, and targets of UbE3A in Angelman Syndrome.

Angelman Syndrome (AS) is a neurological syndrome which causes patients to be excessively cheerful, giggling, and happy, and mentally retarded with very little or no language. AS occurs when the protein ubiquitin E3A ligase (UbE3A) is lost due to abnormal methylation/imprinting of a region of chromosome 15. UbE3A recognizes proteins and attaches a ubiquitin to them, which causes the proteins to be degraded in the proteasome. Loss of UbE3A in Angelman Syndrome would prevent these proteins from being degraded and lead to an excess of them in cells. In the previous post, I described the known functions of the ubiquitination targets of UBE3A. The three most relevant targets are 1) Arc – a protein which regulates synaptic strengths in response to neuronal activity;  2) Ephexin5 – a rho-GEF which regulates cytoskeletal remodeling to restrict synapse number;  and 3)Sacsin – A proteasome-related protein which is important in large neurons of the motor system.

Although the advances in our molecular and genetic understanding of Angelman Syndrome (AS) are commendable, what these changes mean for organ systems and the organism is not obvious. This disconnect is not unique to AS pathophysiology. It is problematic for investigators studying all neurobiological diseases to link identified molecular lesions with neural and behavioral dysfunction. To alleviate this situation, investigators study animal models of disease at the systems (in this case, the nervous system) level.

Fortunately for those interested in AS, a heterozygous ubiquitin E3A (UbE3A) knockout inherited from the mother recapitulates a neurodevelopmental disorder approximating AS in mice (Jiang et al 1998). These mice exhibit motor dysfunction, sound-induced seizures, an abnormal EEG (large amplitude 3 Hz spike wave activity), defective contextual fear-conditioning, and impaired hippocampal LTP. The brain appears normal with standard neuroanatomical and histological techniques. P53 levels are very high in the cells of these mice, alluding to the possibility that UbE3A can ubiquitinate p53 even in the absence of the viral E6 protein (see previous, molecular biology). Miura et al noted mostly the same pattern of findings in their independently-generated UbE3A maternal-KO mouse, but did not find increased p53 levels (2002).

Dindot et al engineered mice to express yellow fluorescent protein (YFP) fused to UbE3A in neurons (2008). In these mice, it was shown that UbE3A localizes to synapses and the nucleus, and that while neurons only use the maternal allele, glia use both the paternal and maternal allele of UbE3A (see genetics). Dendritic arbors were simplified in these mice, with abnormally small hypervariable spines and decreased spine density (Dindot et al 2008). Abnormal synchronous (epilepsy-like) oscillations in the cerebellum of these mice are rescued by gap junction blockers, suggesting that UbE3A may regulate the electrical coupling between cells (Cheron et al 2005). It was recently discovered that these mice also have decreased neurogenesis in the dentate gyrus of the hippocampus, which could have implications for the abnormal learning and LTP in these mice (Mardirossian et al 2009).

Yashiro et al analyzed the visual cortex of AS mice in greater detail (2009). In whole cell recordings from L2/3 pyramidal neurons, they noted a decreased frequency of miniature excitatory postsynaptic potentials (mEPSCs) compared to wild-type (WT) with no change in mEPSC amplitude. The authors also noted plasticity abnormalities in several paradigms. Dark rearing (raising animals in darkness), for example, decreased mEPSC frequency in WT but not in the KO. In addition, decreased long-term potentiation (LTP) and long term depression (LTD) were identified at the L4 to L2/3 synapse, a decrease which progressed in adulthood. Interestingly, LTP and LTD could be induced in dark-reared animals; it was not until normal sensory experience occurred that plasticity was impaired. Furthermore, plasticity could be restored by dark-rearing the mice in adulthood. Monocular deprivation experiments like that described in the previous section for Arc KO mice revealed an identical pattern – no ocular dominance reorganization in favor of the spared eye occurred in AS mice.

UbE3A-maternal-KO mice demonstrate increased levels of inhibitory phosphorylation on CaM kinase II in neurons (Weeber et al 2003). Prevention of this inhibitory phosphorylation by mutation of CaMKII’s threonines 305 and 306 rescues the mouse’s neurological deficits (van Woerden et al 2007). This surprising result is especially relevant given the recent finding that the activity-dependent translocation of proteasomes into dendritic spines depends on CaMKIIα acting as a scaffolding protein (Bingol et al 2010). This activity-dependent translocation is abolished by the T305/306 mutation. From these findings we can formulate this model: In AS, proteasomes translocate to the dendritic spine normally, but important degradation targets like Arc and Ephexin5 are not ubiquitinated and do not enter the proteasome. Some proteins are degraded while UbE3A’s targets remain, which by some mechanism leads to the inhibitory phosphorylation of CamKIIα. This phosphorylation encourages more proteasome to enter the spine, which leads to more aberrant degradation. This vicious cycle produces long, thin, abnormal spines (Dindot et al 2008) which cannot change in response to experience (Yashiro et al 2009). Prevention of CaMKIIα’s inhibitory phosphorylation breaks this cycle by suppressing the translocation of proteasomes into synapses. With fewer spine proteasomes, the UbE3A non-targets are protected from degradation relative to UbE3A targets, and a proteomically-balanced synapse is restored.

Bingol B, Wang CF, Arnott D, Cheng D, Peng J, Sheng M. (2010) “Autophosphorylated CaMKIIalpha acts as a scaffold to recruit proteasomes to dendritic spines.” Cell. 140(4):567-78.

Cheron G, Servais L, Wagstaff J, Dan B. (2005) “Fast cerebellar oscillation associated with ataxia in a mouse model of Angelman syndrome.” Neuroscience 130:631–637.

Dindot SV, Antalffy BA, Bhattacharjee MB, Beaudet AL. (2008) “The Angelman syndrome ubiquitin ligase localizes to the synapse and nucleus, and maternal deficiency results in abnormal dendritic spine morphology.” Hum Mol Genet 17:111–118.

Jiang, Y., Armstrong, D., Albrecht, U., Atkins, C. M., Noebels, J. L., Eichele, G., Sweatt, J. D., & Beaudet, A. L. (1998) “Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation.” Neuron 21, 799–811

Mardirossian S, Rampon C, Salvert D, Fort P, Sarda N. (2009) “Impaired hippocampal plasticity and altered neurogenesis in adult Ube3a maternal deficient mouse model for Angelman syndrome.” Exp Neurol. 220(2):341-8.

Miura K, Kishino T, Li E, Webber H, Dikkes P, Holmes GL, Wagstaff J. (2002) “Neurobehavioral and electroencephalographic abnormalities in Ube3a maternal-deficient mice” Neurobiol Dis 9:149–159.

van Woerden, G.M., Harris, K.D., Hojjati, M.R., Gustin, R.M., Qiu, S., de Avila Freire, R., Jiang, Y.H., Elgersma, Y. and Weeber, E.J. (2007) “Rescue of neurological deficits in a mouse model for Angelman syndrome by reduction of alphaCaMKII inhibitory phosphorylation.” Nat. Neurosci., 10, 280–282.

Weeber, E.J., Jiang, Y.H., Elgersma, Y., Varga, A.W., Carrasquillo, Y., Brown, S.E., Christian, J.M., Mirnikjoo, B., Silva, A., Beaudet, A.L. et al. (2003) “Derangements of hippocampal calcium/calmodulin-dependent protein kinase II in a mouse model for Angelman mental retardation syndrome.” J. Neurosci., 23, 2634–2644.

Yashiro, K., et al., Ube3a is required for experience-dependent maturation of the neocortex. Nat Neurosci, 2009. 12(6): p. 777-783.