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. 2018 Oct 19;9:54.
doi: 10.1186/s13229-018-0238-0. eCollection 2018.

Subcellular Organization of UBE3A in Human Cerebral Cortex

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Free PMC article
Case Reports

Subcellular Organization of UBE3A in Human Cerebral Cortex

Alain C Burette et al. Mol Autism. .
Free PMC article

Abstract

Background: Loss of UBE3A causes Angelman syndrome, whereas excess UBE3A activity appears to increase the risk for autism. Despite this powerful association with neurodevelopmental disorders, there is still much to be learned about UBE3A, including its cellular and subcellular organization in the human brain. The issue is important, since UBE3A's localization is integral to its function.

Methods: We used light and electron microscopic immunohistochemistry to study the cellular and subcellular distribution of UBE3A in the adult human cerebral cortex. Experiments were performed on multiple tissue sources, but our results focused on optimally preserved material, using surgically resected human temporal cortex of high ultrastructural quality from nine individuals.

Results: We demonstrate that UBE3A is expressed in both glutamatergic and GABAergic neurons, and to a lesser extent in glial cells. We find that UBE3A in neurons has a non-uniform subcellular distribution. In somata, UBE3A preferentially concentrates in euchromatin-rich domains within the nucleus. Electron microscopy reveals that labeling concentrates in the head and neck of dendritic spines and is excluded from the PSD. Strongest labeling within the neuropil was found in axon terminals.

Conclusions: By highlighting the subcellular compartments in which UBE3A is likely to function in the human neocortex, our data provide insight into the diverse functional capacities of this E3 ligase. These anatomical data may help to elucidate the role of UBE3A in Angelman syndrome and autism spectrum disorder.

Keywords: Angelman syndrome; Axon terminal; E6-AP; Euchromatin; Mitochondria.

Conflict of interest statement

All procedures regarding human tissue were performed with the approval of the UCSF Committee on Human Research and the University of Maryland Institutional Review Board.All authors read and approved the final manuscript.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Ultrastructural integrity of human tissue samples from different sources. Ultrastructural integrity is low in postmortem material from a control donor, though major organelles such as nuclei (N) remain identifiable (low-magnification view, a). At high magnification, synapses (arrowhead) are damaged but clearly defined (b). Material from an Angelman syndrome donor is virtually uninterpretable (reflecting the practical difficulty of obtaining and curating such material), though damaged mitochondria and traces of nuclear structure remain (N in panel c); hints of synaptic specializations may be identified even in this material (arrowhead, d). In contrast, tissue from biopsy specimens obtained during surgeries for epilepsy shows very good preservation, as documented by features including intact mitochondria, well-aligned rough endoplasmic reticulum, and intact chromatin (e). The well-preserved neuropil (f) contrasts with that of the autopsy specimens above. Tissue from case no. 5758 for a and b, 1494 for c and d, and Q1012 for e and f. Scale bar = 1 μm in a, c, and e and 500 nm in b, d, and f
Fig. 2
Fig. 2
Immunohistochemistry for UBE3A in mouse cerebral cortex following different methods of fixation. UBE3A staining in tissue fixed by perfusion (a, b). c, d Immersion fixation of mouse cortex after a delay of 15 min (similar to the fixation for our surgical material) shows only modest differences, including a subtle condensation of immunopositive cytoplasm. In both cases, UBE3A is expressed through all layers of the neocortex, with strong nuclear staining and weaker staining in somata and dendrites. UBE3A staining is nearly absent in the brain of an AS model mouse fixed by immersion after a delay of 15 min (e, f), further confirming specificity of the UBE3A antibody in brain tissue harvested under conditions similar to surgical biopsy. Scale bar = 100 μm in a, c, and e and 25 μm in b, d, and f
Fig. 3
Fig. 3
UBE3A expression in temporal cortex from a human biopsy specimen. a Immunoperoxidase staining for UBE3A. As can be seen by comparison with adjacent Nissl-stained section (b), the large majority of neurons are immunopositive. c Enlargement of boxed area from a; UBE3A is expressed throughout all layers of temporal cortex. d, e Higher magnification micrographs show UBE3A concentrating in nuclei. Antigen is also visible in somata and neuropil, but at lower levels. f High magnification micrograph of a 250-nm plastic section shows discrete grains of immunoperoxidase reaction product scattered through most of the nucleus, but seemingly excluded from certain regions. Tissue from case no. Q1019. Scale bar = 1 mm in a and b, 250 μm in c, 50 μm in d, 25 μm in e, and 10 μm in f
Fig. 4
Fig. 4
UBE3A expression in GABAergic neurons in temporal cortex from a human biopsy specimen. a Double labeling reveals UBE3A in the nuclei and somata of GABAergic neurons (arrows). b A pyramidal neuron immunopositive for UBE3A is surrounded by numerous GABA-positive structures likely to represent cross sections of dendrites and axon terminals. Enlargements in c and d show the presence of UBE3A in some of these small GABA-positive structures. Tissue from case no. Q1010. Scale bar = 50 μm in a, 10 μm in b, and 2 μm in c and d
Fig. 5
Fig. 5
UBE3A expression in neuroglia in temporal cortex from human biopsy specimen. a, b Double labeling reveals UBE3A in the nuclei and somata of Olig2-positive glia (oligodendrocytes, arrowheads). However, UBE3A staining in Olig2-positive glia (b, arrowhead) is appears weaker than in neurons (b, arrow). c Double labeling reveals UBE3A in the nuclei and somata in GFAP-positive cells (astrocytes). UBE3A in GFAP-positive cells is at lower levels than in neurons and Olig2-positive glia. Tissue from case no. Q1010. Scale bar = 50 μm in a, 10 μm in b and c, and 5 μm in d
Fig. 6
Fig. 6
High magnification immunofluorescence, showing UBE3A (green) in two pyramidal neurons in layer V in temporal cortex from human biopsy specimen (a). Sections were counterstained with DAPI (red) to visualize nuclei. UBE3A staining is organized into small puncta that concentrate in neuronal nuclei. Small UBE3A-positive puncta are visible in somata and proximal dendrites, and throughout the neuropil. Successive enlargements (b, c) show that UBE3A puncta are excluded from the nucleolus (Nu) and from DAPI “hotspots.” Scale bar = 10 μm in a, 5 μm in b, and 1 μm in c. Tissue from case no. Q1019
Fig. 7
Fig. 7
Quantification of the reciprocal localization of UBE3A and DAPI in human neuronal nuclei. To analyze the relationship between UBE3A and DAPI in neuronal nuclei, we used the intensity correlation analysis approach of Li et al. [47]. Analyses are presented as intensity correlation plots: the x value, (channel 1 pixel value–channel 1 mean value) × (channel 2 value–channel 2 mean value), reflect the covariance of both channels, and the y value reflects the intensity of channels 1 or 2. Pixels with values situated left of the x = 0 line do not colocalize or have inversely correlated intensities, whereas pixels situated on the right side colocalize (see diagram in a). Scatterplot in b and c corresponds to the nuclear region of the sections used for illustration in Fig. 6b (Scatterplots are from raw confocal images, while contrast and brightness were adjusted in the micrographs). Panel c shows DAPI with respect to UBE3A, and d shows UBE3A with respect to DAPI. Both plots are skewed toward negative values, implying that UBE3A and DAPI pixel intensity co-varies in opposite directions. d Box and whiskers plot of intensity correlation quotient (ICQ, [47]) from 92 nuclei from layers II–III. ICQ values ~ 0 imply random staining, 0 > ICQ ≥ − 0.5 indicate segregated (negatively correlated) staining, and 0 < ICQ ≤ + 0.5 indicate dependent (positively correlated) staining. We found an average ICQ of − 0.04 ± 0.005, showing that UBE3A and DAPI staining negatively co-vary
Fig. 8
Fig. 8
Pre-embedding immunogold labeling for UBE3A in layer II/III lateral temporal cortex from human biopsy specimen. a, b UBE3A labeling in nucleus (colorized in blue); labeling is over euchromatin (Ec) domains, but is not associated with heterochromatin (Hc) or nucleoli (Nu). c UBE3A labeling associated with endomembranes of the Golgi apparatus. d UBE3A labeling in cytoplasm; arrows point to label associated with mitochondria. e, f UBE3A labeling in presynaptic terminals (colorized in green) and a dendritic spine (colorized in red). g A GABAegic terminal (colorized in yellow) labeled for UBE3A (pre-embedding, irregular 30–50 nm particles of silver-intensified immunogold, arrowheads) and GABA (post-embedding, 20 nm gold). Tissue from case no. Q1010 in a, c, d, e, and f and Q1019 in b and g. Scale bar = 1 μm in a and b, 250 nm in c and g, and 500 nm in df

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