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

Grier MD, Carson RP, Lagrange AH - PLoS ONE (2015)

Bottom Line: Utilizing enriched primary cultures of oligodendrocytes and astrocytes, we show that Ube3a is expressed, but not imprinted in these cell types.To determine the temporal expression pattern and silencing, we analyzed Ube3a expression in AS mice at several time points.We confirm relaxed imprinting of Ube3a in neurons of the postnatal developing cortex, but not in structures in which neurogenesis and migration are more complete.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Vanderbilt University Medical Center, Nashville, TN 37232-8552, United States of America.

ABSTRACT
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 Ube3a gene. Ube3a 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 Ube3a 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 Ube3a 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 Ube3a 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.

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Ube3a is expressed, but not imprinted in cultured astrocytes from AS animals.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.
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pone.0124649.g002: Ube3a is expressed, but not imprinted in cultured astrocytes from AS animals.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.

Mentions: A growing body of clinical evidence has suggested that myelin dysfunction may be a contributing factor to morbidity in a variety of neurodevelopmental syndromes[16–18]. Recent diffusion tensor imaging (DTI) studies have found altered white matter tracts, thinned corpus callosa and delayed myelination and in Angelman patients[19–21]. While earlier studies suggest that Ube3a imprinting occurs primarily in neurons, recent evidence suggests Ube3a is not imprinted in glial cells [12,22]. In addition, recent work from our lab has revealed disrupted myelin protein expression in AS mice, thus we sought to determine the extent to which Ube3a is expressed in glial cells and whether the expression is maternally imprinted. Expression of Ube3a was determined in primary astrocyte and oligodendrocyte cultures derived from WT and AS mouse cortex. Immunofluorescent micrographs of glial fibrillary acid protein (GFAP) positive astrocytes from WT mice demonstrated intense nuclear and diffuse cytosolic staining for Ube3a (Fig 2, upper panels). While the cultures were highly enriched, Ube3a immunoreactivity could occasionally also be noted in GFAP-negative cells. Astrocytes derived from AS mice showed a reduction in Ube3a immunoreactivity compared to WT astrocytes, but not the complete loss that would be expected were it imprinted (Fig 2, lower panels). To better quantitate the difference in expression, protein lysates from the enriched astrocyte cultures were analyzed by Western blot and show a reduction of Ube3a of approximately 50% compared to WT control (Fig 3). This is consistent with biallelic expression and supports that Ube3a is not imprinted in astrocytes.


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

Grier MD, Carson RP, Lagrange AH - PLoS ONE (2015)

Ube3a is expressed, but not imprinted in cultured astrocytes from AS animals.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403805&req=5

pone.0124649.g002: Ube3a is expressed, but not imprinted in cultured astrocytes from AS animals.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.
Mentions: A growing body of clinical evidence has suggested that myelin dysfunction may be a contributing factor to morbidity in a variety of neurodevelopmental syndromes[16–18]. Recent diffusion tensor imaging (DTI) studies have found altered white matter tracts, thinned corpus callosa and delayed myelination and in Angelman patients[19–21]. While earlier studies suggest that Ube3a imprinting occurs primarily in neurons, recent evidence suggests Ube3a is not imprinted in glial cells [12,22]. In addition, recent work from our lab has revealed disrupted myelin protein expression in AS mice, thus we sought to determine the extent to which Ube3a is expressed in glial cells and whether the expression is maternally imprinted. Expression of Ube3a was determined in primary astrocyte and oligodendrocyte cultures derived from WT and AS mouse cortex. Immunofluorescent micrographs of glial fibrillary acid protein (GFAP) positive astrocytes from WT mice demonstrated intense nuclear and diffuse cytosolic staining for Ube3a (Fig 2, upper panels). While the cultures were highly enriched, Ube3a immunoreactivity could occasionally also be noted in GFAP-negative cells. Astrocytes derived from AS mice showed a reduction in Ube3a immunoreactivity compared to WT astrocytes, but not the complete loss that would be expected were it imprinted (Fig 2, lower panels). To better quantitate the difference in expression, protein lysates from the enriched astrocyte cultures were analyzed by Western blot and show a reduction of Ube3a of approximately 50% compared to WT control (Fig 3). This is consistent with biallelic expression and supports that Ube3a is not imprinted in astrocytes.

Bottom Line: Utilizing enriched primary cultures of oligodendrocytes and astrocytes, we show that Ube3a is expressed, but not imprinted in these cell types.To determine the temporal expression pattern and silencing, we analyzed Ube3a expression in AS mice at several time points.We confirm relaxed imprinting of Ube3a in neurons of the postnatal developing cortex, but not in structures in which neurogenesis and migration are more complete.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Vanderbilt University Medical Center, Nashville, TN 37232-8552, United States of America.

ABSTRACT
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 Ube3a gene. Ube3a 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 Ube3a 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 Ube3a 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 Ube3a 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.

Show MeSH
Related in: MedlinePlus