Ube3a-like immunoreactivity (Ube3a-li) is absent throughout the brain in AS mice. Coronal sections through cortex, hippocampus, thalamus, and brainstem are shown. Ube3a-li is seen in cells throughout the brain of WT mice (left), while no specific Ube3a-li is detected in the AS mouse brain (right). Scale bars are 2000 μm for the top panels and 250 μm for the magnified panels below.

Distribution of Ube3a-like immunoreactivity (Ube3a-li) in the mouse brain. A. Dense Ube3a-li neurons are seen in the pyramidal cell layer and in scattered cells of the stratum oriens of the dorsal hippocampus from a WT mouse. B. No Ube3a-li elements are present in the dorsal hippocampus of the AS mouse. C. Ube3a-li cells are seen throughout the thalamus, with particularly dense staining seen in the cells of the paraventricular thalamic nucleus. D. In the somatosensory and other cortices, Ube3a-li cells can be seen in layers II-VI. E. Ube3a-li cells are seen through the dorsal striatum. F. Ube3a-li marks cells of the amygdala complex, with prominent staining seen in the central and especially medial nuclei, and what appears to be a somewhat lower density of cells in basolateral nucleus. G. Cells immunoreactive for Ube3a are present it the dorsal pons and overlying cerebellar cortex; in the pons, Ube3a-li cells can be seen in the locus ceruleus, parabrachial nucleus, and dorsal tegmental nucleus. H. Densely packed Ube3a-li cells in the substantia nigra and medially contiguous ventral tegmental nucleus are present, with fewer Ube3a-li cells seen in the pars reticulata. Scale bars are 500 μm C and 200 μm (F-H); the scale bar in G also applies to panels A, B, D, and E.

Ube3a-li is present in interneurons but not astrocytes. A. In the neocortex some parvalbumin(PV)-li interneurons (brown) exhibiting nuclear labeling for Ube3a-li (arrow) are seen next to cells that are express only Ube3a-li (black) or parvalbumin (arrowhead) but not Ube3a. B. A calretinin-li cell expressing Ube3a-li (arrow) is seen dorsal to a dense band of Ube3a-li positive hippocampal dentate granule cells. C. In the thalamic reticular nucleus the great majority of cells show both PV- and Ube3a-li (arrow). D. Astrocytes, which express the GFAP-li (brown), do not express Ube3a-li (seen in black IR nuclei) in the hippocampus. Scale bars are 25 μm in panel C and 10 μm in panel D; the scale bar in panel D applies also to panels A and B.

Subcellular Ube3a expression pattern in hippocampal neuron cultures. Ube3a is ubiquitously expressed throughout rat hippocampal neurons, with the most intense staining levels in the nucleus, but also with prominent staining in the cytoplasm and dendrites. A. Ube3a-li partially co-localizes with the presynaptic marker synapsin. B. Ube3a-li and PSD-95-li partially co-localize in dendrites of rat hippocampal neurons. Regions of co-localization are depicted in yellow. Scale bars are 20 μm for upper panels and 5 μm for lower panels.

Ube3a expression in peripheral tissues is decreased in AS mice. Western blot analysis of total homogenates from heart, liver, and kidney of WT and AS mice demonstrates that Ube3a is expressed in these peripheral tissues. Quantification of Western blots (top row) were normalized to the Ponceau-S total protein stain and set as a percentage of WT (graphs below Western blots). Ube3a expression levels in heart, liver and kidney of AS mice are reduced by ≈70% compared to corresponding tissues from WT mice. Animals: WT N=3, AS N=4.

Ube3a expression in brain is dramatically reduced in AS mice. A. Western blot analysis of total forebrain homogenates from WT (M+/P+), AS (M−/P+), UBE3A M+/P−, and UBE3A knockout (M−/P M−) mice shows a loss of Ube3a protein in AS mice compared to that of WT and AS mice when probing with the Sigma-Aldrich Ube3a antibody. Overexposure of the Western blot (bottom blot in A) reveals low levels of specific Ube3a staining in AS mice, as compared with the complete loss of expression in Ube3a knockout (M−/P −) mice. Arrows correspond to specific Ube3a staining that is not seen in M −/P − mice. B. Western blot analysis of total brain homogenates from hippocampus, striatum, hypothalamus, thalamus, cortex, cerebellum, midbrain, and olfactory bulbs reveals an estimated 90% loss of Ube3a expression in AS mice compared to WT mice (also see Supplementary Figure 3). C. Relative Ube3a expression across brain regions of WT mice quantified from Western blots in panel A, by normalizing to Ponceau-S total protein stain, shows the highest expression in the cortex, thalamus, and olfactory bulbs with lowest expression in the striatum. Animals: WT N=3, AS N=4.

Ube3a localizes to multiple subcellular fractions. Subcellular fractionation and Western blot analysis of hippocampal homogenates shows Ube3a co-fractionates strongly with the cytoplasmic protein, GAPDH. Ube3a is co-fractionated to a lesser extent with the endoplasmic reticulum associated protein, IP3R (membrane-associated fraction), and with PSD-95 (postsynaptic density-associated fractions).