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 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

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

Multi-organ Abnormalities and mTORC1 Activation in Zebrafish Model of Multiple Acyl-CoA Dehydrogenase Deficiency

<div><p>Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the <i>ETFA</i>, <i>ETFB</i>, and <i>ETFDH</i> genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant <i>dark xavier</i> (<i>dxa^vu463</i>) that has an inactivating mutation in the <i>etfa</i> gene. <i>dxa^vu463</i> recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant <i>dxa^vu463</i> zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant <i>dxa^vu463</i> zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that <i>etfa</i> gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.</p></div

Facial axons, mechanosensory hair cell and myelination defects in type II <i>dxa^vu463</i> mutant zebrafish.

<p>(A) DIC imaging of live cilia (first column) and neuromast cells (second column, asterisks), ORO stained lipids in neuromast cells (third column, asterisks). Acetylated-tubulin marks cilia (yellow arrowheads). Magnified views of cilia are shown on the upper right corner. (B) Whole mount immunofluorescence staining of acetylated-tubulin in WT (left) and <i>dxa</i> mutants (right) at 8 dpf. Yellow arrowheads indicate facial axons. Rectangle region is magnified in lower left corner. (C) anti-MBP staining in the brain (left) and spinal cord (right) in WT (top) and <i>dxa</i> mutant zebrafish (bottom) at 8 dpf. Arrows indicate myelinated axons in the spinal cord, all signal is reduced in <i>dxa</i> mutant zebrafish. (D) TEM (11,000×) image of spinal cord as indicted by arrows in C. Normal (WT) and swollen (<i>dxa</i> mutant) mitochondria are pseudocolored green, indicated by large red arrowhead). Red arrows indicate less condensed myelination layer in a <i>dxa</i> axon. Further magnified views of mitochondria are on the lower left corner. M, Mauthner axon track. Scale bars are as indicated in each panel.</p

Tissue dependent regulation of mTORC1 activation in <i>dxa^vu463</i> mutant zebrafish.

<p>Anti-phospho-S6 (left panels) and anti-phospho-4E-BP1 (right panels) antibodies were used to assess mTORC1 kinase activity. (A) WT brain (top) and <i>dxa</i> brain (bottom) at 8 dpf. Arrows indicate p-S6 and phospho-4E-BP1 positive cells in neural progenitors of the brain. P-S6 and p-4E-BP1 are also detected in the superficial pial cells of the mutant brain. (B) Sections of trunk regions in WT (top) and <i>dxa</i> (bottom) at 8 dpf. Phospho-S6 was detected in the central canal and phospho-4E-BP1 positive cells were found central canal as well as midline cells (yellow arrow) in <i>dxa</i> mutant zebrafish. Asterisks indicates central canal of hindbrain. 300 nM of rapamycin was used from 5 dpf to 8 dpf to treat <i>dxa</i> mutant zebrafish in C and D. (C) Hindbrain regions of type III mutant at 8 dpf. Phospho-S6 and phospho-4E-BP1 staining was again detected in central canal (*) and pial cell sheath (P) in both control and rapamycin treated <i>dxa</i> mutants. DAPI (blue) was used for nuclei staining. (D) Liver regions of same sections seen in (C) with marked suppression of phospho-S6 but a relative increase in phsohp-4E-BP1 levels. P, pial cell sheath; HB, hindbrain; K, kidney; L, liver. Scale bar = 100 µm.</p

Lipids, cerebroside sulfate and free cholesterol accumulations in the cytosol of type II <i>dxa^vu463</i> hepatocyte.

<p>(A) Whole mount Oil Red-O (ORO) staining of wild type and type II <i>dxa</i> at 8 dpf. Vertical line (a) indicates location of transverse section in B. (B) ORO staining in the liver sections at 8 dpf. (C) Toluidine blue, PAS and Filipin staining at 9 dpf. Wild type control livers are on the top row and <i>dxa</i> are on the bottom row. Magnified views of rectangles showing toluidine blue are in the lower left corners. The brown colored drops in Toluidine blue staining suggests cerebroside sulfate accumulation. Magnified views of rectangles showing Filipin (free cholesterol) staining are in the upper right corners. Filipin appears to accumulate in the cytosol of mutant hepatocytes. (D) TEM image at 6 dpf. Green shadows mark single representative mitochondria. Magnified views of rectangles showing cristae are on the left lower corners. (E) TEM image at 8 dpf. Nuclei are colored red and single representative mitochondria are again colored green. Dark granules in <i>dxa</i> mutants appear to represent lipid drops. Mib, midbrain; I, intestine; L, liver; bv, blood vessels; N, nucleus; cyto, cytosol; Mito, mitochondria; Lip, lipid drops. Scale bar = (A) 100 µm, (B) 50 µm, (C) 25 µm and (D, E) 2 µm.</p

Classification of <i>dxa^vu463</i> homozygous mutants, positional cloning of <i>dxa^vu463</i> and Etfa protein expression.

<p>(A) Representative phenotypes of most severe (type I), moderate (type II) and mild (type III) <i>dxa</i> homozygous mutants at 7 dpf. Blue lines (a and b) indicate region of transverse sections in D. (B) Spectrum changes of type I, II and III mutants under different feeding conditions. Blue bars indicate the proportion of mutants under regular feeding (n = 218, 5 clutches), red bars for the proportion of mutants under extra feeding condition (n = 151, 3 clutches), p* = 0.03, p** = 0.00015. (C) Primary predicted structure of Etfa protein in wild-type and <i>dxa</i> zebrafish. Shaded codon indicates the null mutation of <i>etfa</i> in <i>dxa</i> zebrafish (GGA (Glycine) to TGA (stop)). (D) Anti-Etfa immunostaining (red) in wild-type control (upper panel, n = 9/9) and homozygous mutant (lower panel, n = 9/9) at 9 dpf. DAPI (blue) was used for nucleus staining. Arrows indicate Etfa expression in the ventricular region of the brain. Magnified midline views of yellow rectangles are in the left corners. Magnified rectangles on the trunk sections indicate neuromast hair cells. NM, neuromast; PF, pectoral fin; K, kidney; L, liver. Scale bar = 100 µm.</p