Toward a Broader View of Ube3a in a Mouse Model of Angelman Syndrome: Expression in Brain, Spinal Cord, Sciatic Nerve and Glial Cells

<div><p>Angelman Syndrome (AS) is a devastating neurodevelopmental disorder characterized by developmental delay, speech impairment, movement disorder, sleep disorders and refractory epilepsy. AS is caused by loss of the Ube3a protein encoded for by the imprinted <i>Ube3a</i> gene. <i>Ube3a</i> is expressed nearly exclusively from the maternal chromosome in mature neurons. While imprinting in neurons of the brain has been well described, the imprinting and expression of Ube3a in other neural tissues remains relatively unexplored. Moreover, given the overwhelming deficits in brain function in AS patients, the possibility of disrupted Ube3a expression in the infratentorial nervous system and its consequent disability have been largely ignored. We evaluated the imprinting status of <i>Ube3a</i> in the spinal cord and sciatic nerve and show that it is also imprinted in these neural tissues. Furthermore, a growing body of clinical and radiological evidence has suggested that myelin dysfunction may contribute to morbidity in many neurodevelopmental syndromes. However, findings regarding Ube3a expression in non-neuronal cells of the brain have varied. Utilizing enriched primary cultures of oligodendrocytes and astrocytes, we show that <i>Ube3a</i> is expressed, but not imprinted in these cell types. Unlike many other neurodevelopmental disorders, AS symptoms do not become apparent until roughly 6 to 12 months of age. To determine the temporal expression pattern and silencing, we analyzed Ube3a expression in AS mice at several time points. We confirm relaxed imprinting of <i>Ube3a</i> in neurons of the postnatal developing cortex, but not in structures in which neurogenesis and migration are more complete. This furthers the hypothesis that the apparently normal window of development in AS patients is supported by an incompletely silenced paternal allele in developing neurons, resulting in a relative preservation of Ube3a expression during this crucial epoch of early development.</p></div

Ube3a expression time course in cortical lysates.

<p>A). Representative data for expression of Ube3a in P0, P3, P6 and P42 cortical lysates. B) Quantification of Ube3a expression at various time points. P0 cortical lysates express approximately 25% of WT protein, compared to approximately 5% of WT expression at P3 and later. *** indicates P ≤.0001 by a one-way ANOVA with Tukey's multiple comparison test comparing AS animals at each time point. n = 7–10 per group.</p

Ube3a is expressed, but not imprinted in cultured astrocytes from AS animals.

<p>A) WT upper panels, AS lower panels. Left to right: GFAP (a marker for astrocytes), Ube3a and merge of GFAP with Ube3a and DAPI. Ube3 expression is most apparent in the nucleus of GFAP positive cells, with lower levels of expression throughout the cytosol. DAPI colocalizes with nuclear Ube3a. Scale bar represents 200 μm.</p

Ube3a is expressed, but not apparently imprinted in cultured oligodendrocytes from AS animals.

<p>WT upper panels, AS, lower panels. Left to Right: Myelin Basic Protein (MBP, a marker for oligodendrocytes), Ube3a and merge with DAPI. Ube3a is expressed throughout oligodendrocytes as shown by robust colocalization with MBP in both WT and AS oligodendrocytes. Arrows highlight Ube3a (+), MBP (-) cells, likely to be contaminating astrocytes. Scale bar represents 100 μm.</p

Ube3a expression in other brain regions

<p>A) Representative data for Ube3a expression in P0 and P42 subcortical lysates (thalamus and hypothalamus). B) Quantification shows approximately 5–10% residual paternal Ube3a at birth and P42. c) Representative data for Ube3a expression in cerebellar lysates. d) Quantification shows 10–15% residual paternal Ube3a at P0 and P42. N = 3–5 per group.</p

P15 APC P5 PDGFRa images

Images for APC and PDGFRa cell count

Western Blots

Western Blot

Rictor CKO Western

Rictor CKO Wester

Barnes Maze

Barnes Maz

P60 APC images

APC images used for P60 cell count