Introduction
Angelman syndrome (AS) is a genetic disorder with an incidence of 1 in 15,000, and it was first described in 1965 by Harry Angelman (1,2). It is characterized by a severe developmental delay together with mental disorders, movement disorders and behavioral abnormalities. Early severe epilepsy, sleep alteration, ataxia, important gait, absence of language and craniofacial dysmorphism are phenotypic characteristics used as diagnostic criteria of AS (3).
Several genetic mechanisms are known to associate with the development of Angelman syndrome including the deletion of 4 Mb region in chromosome 15q11-13, uniparental disomy (UPD), imprinting centre defects, and mutation in UBE3A (4). The loss of expression of imprinted genes causes multiple human genetic disorders, including AS and Prader-Willi syndrome (PWS). Although these two diseases are associated with the lack of gene expression from the same chromosome 15q11-q13 region, the clinical features of the two disorders are distinct. Deletion or loss of paternally inherited gene expression results in PWS, while loss of maternally inherited gene expression causes AS (4).
Multiple mouse models have been developed for the study of AS (Table 1). The first reported AS mouse model generated was a mouse with paternal UPD for chromosome 7 (5), followed by another mouse model generated by radiation-induced deletion of p locus and Ube3a (6). However, these two models carried a large deletion of mouse chromosome 7C that could affect multiple loci (1). In the current study, we used a mouse model which carried an exon 2 deletion of the Ube3a gene resulting in a shift in the reading frame, thereby inactivating all putative isoforms of Ube3a (7). If the offspring mice inherited the mutated Ube3a allele of maternal origin, the mice will have no Ube3a expression in the cerebellum, Purkinje cells and hippocampus, as Ube3a on the paternal chromosome is silenced by genomic imprinting. This mouse model exhibits symptoms similar to that of Angelman syndrome patients, including motor dysfunction, seizures, context-dependant learning deficiency and severely impaired long-term potentiation (LTP) (7).
Several proteins that are involved in REDOX (oxidation-reduction reactions) were identified in the 2-D DIGE, including LDH, MDH, GSTs-Mu1, SOD2, and ATP5a1. The result suggested that loss of Ube3a may lead to mitochondrial dysfunction. In addition, the accumulation of Chaperone protein Hsp70 was observed and mRNA levels remained unchanged, suggesting that Hsp70 might be a substrate of Ube3a. Furthermore, NSF, which is known to be involved in neuronal signal transmission, was reduced at protein levels but unaffected at mRNA levels. Finally, CaBP is responsible for binding free calcium ions and may play an inductive role in seizures observed in AS mouse models and patients. TPI1, Triosephosphate isomerase 1, is one of the key enzymes in the glycolysis pathway, while CFL1, Cofilin 1, is known to be a potent regulator of actin filament dynamics. It remains to be determined how differential expression of these proteins may contribute to the development of AS.
|
|
Mutation |
Phenotype |
Reference |
|
1 |
Ube3a exon 2 deletion |
AS |
(7) |
|
2 |
LacZ insertion inactivation of Ube3a |
AS |
(8) |
|
3 |
insertion/duplication located 13 kb upstream of Snrpn exon 1 |
AS imprinting mutation |
(9) |
|
4 |
80-kb deletion located upstream of Snrpn exon 1 |
AS imprinting mutation |
(9) |
|
5 |
Ube3a-Gabrb3 -Atp10a deletion |
AS |
(10) |
|
6 |
Replacement of mouse PWS-IC with human PWS-IC |
PWS and AS imprinting mutation |
(11) |
|
7 |
UPD |
AS |
(5) |
|
8 |
GABRB3 inactivation |
 |
(12) |
|
9 |
Transgenic insertion induced deletion; Zfp127-Herc2 deletion |
PWS/AS |
(13) |
Table 1. Angelman syndrome mouse models
Materials and methods
Protein extraction
Tissue was homogenized in extraction buffer containing 7 M Urea (Cat. No. U5128, Sigma), 2 M Thiourea (Cat. No. RPN 6301, Amersham), 30 mM Tris (Cat. No. 75825, USB), 4% CHAPS (Cat. No. 13361, USB), adjusted to pH 8.5 with HCl. Complete protease inhibitor cocktail (Cat. No. 1697498, Roche) and nuclease mix (Cat. No. 80-6501-42, Amersham) were added into extraction buffer before use. Tissue was homogenized with 3-s pulses followed by 5-s of cooling on ice between the pulses, until no visible tissue could be observed. The homogenized sample was then transferred to the centrifuge tube and centrifuged at 20,000 x g for 20 min. The supernatant was transferred into a new centrifuge tube and centrifuged for another 20 min at 20,000 x g. The supernatant was then aliquoted and stored at -80°C. The protein concentration was determined by using Bio-Rad protein assay (Cat. No. 500-0002, Bio-Rad) based on Bradford’s method according to the manufacturer’s protocol.
CyDye labelling
Cy2 minimal dye, Cy3 minimal dye, Cy5 minimal dye (Cat. No. 25-8008-60, Cat. No. 258008-61, Cat. No. 25-8008-62, Amersham) are the three cyanine dyes used in the experiment. CyDye was reconstituted by using N-N-Dimethylformamide (Cat. No. 22,705-6, Aldrich). 400 pmol of CyDye was used to label 50 μg of protein as recommended by the manufacturer. The labelling reaction was performed on ice for 30 min, quenched with 1 μ! of 10 mM lysine (Cat. No. L5501, Sigma) and incubated on ice for 10 min. Cy2 was always used to label the internal control as recommended by the manufacturer. Alternative use of Cy3 and Cy5 for the labelling of wild type and diseased samples prevented labelling bias. In the labelling reaction, the ratio of "dye: protein" was kept low to ensure optimal labelling efficiency.
1-D isoelectric focusing
ImmobilineTM Dry strip, pH 3-11NL, 24 cm strip (Cat. No. 17-6003-77, Amersham) was used for the isoelectric focusing. The strip was rehydrated using rehydration buffer containing 8 M Urea, 4% CHAPS, 1% Pharmalyte 3-11 (Cat. No. 17-6004-40, Amersham), 13 mM DTT (Cat. No. 17-1318-02, Amersham), Destreak solution (Cat. No. 71-5025-39 Amersham). Rehydration was done for 16-18 hr. The rehydrated strip was then transferred to a strip holder and placed on the IPGphor (Cat. No. 80-6414-02, Amersham) that was used for isoelectic focusing. The protein lysate was then applied to the strip by cup loading method, and an equal volume of sample buffer (8 M Urea, 130 mM DTT, 4% CHAPS, 2% Pharmalyte 3-11) was added into the labelled protein sample. The protein was focused on 200 Vhr for each 10 μ of sample applied, followed by 500 Vhr, 1000 Vhr, 1000-8000 V gradient increment for 1hr, and 8000 V for 32,000 Vhr. The strip was equilibrated before it was applied to the 2D electrophoresis unit, first with DTT (Cat. No. 17-1318-02, Amersham) in 10 ml equilibration buffer (6 M Urea, 50 mM Tris-Cl, 30% glycerol, 2% SDS, Bromophenol blue; Glycerol Cat. No. 16374, USB) for 20 min and followed with IAA (Cat. No. RPN 6302, Amersham) for another 20 min.
2-D gel electrophoresis
The equilibrated strip was transferred to SDS-PAGE and sealed with 1% agarose sealing solution with bromophenol blue (Cat. No. 12370, USB) as trace dye. Gel electrophoresis was performed on 12% acrylamide SDS-PAGE (40% stock, Cat. No. 17-1310-01, Amersham) casted one night before usage, in 2X SDS running buffer (50 mM Tris, 384 mM Glycine, 0.4% SDS; Glycine Cat. No. 161-0718 Bio-Rad, SDS Cat. No. 75819, USB) at 15°C. 5 W per gel was applied for protein entry, and 10 W per gel for protein separation. The electrophoresis run was stopped when the dye front reached the bottom of the gel. The electrophoresis run was performed on Ettan DALT six system (Cat. No. 80-6485-27, Amersham). The 2-D spot pattern comparison was then made by using Decyder software (Amersham) to figure out protein candidates with significant different steady state levels and those differences in expression were consistent in all 2-D DIGE sample analyzed.
Silver staining proteins visualization
The acrylamide gel was fixed in 50% Methanol (Cat. No. A-454-4, Fisher), 12% Acetic acid (Cat. No. 1.00063.2511, Merck) overnight with mild shaking. The silver staining was performed using Silver Stain Plus kit (Cat. No. 161-0449, Bio-Rad) according to manufacturer’s protocol. The stained gel was then stored in 1% acetic acid solution.
MALDI-TOF protein identification
Stained gel spots were excised by scalpels and cut into 1 mm3 cubes. Silver stained gel spots were then destained by using 100 mM Sodium Thiosulfate (Cat. No. A3525, Applichem) and 30 mM Potassium Ferricyanide (III) (Cat. No. 24.402-3, Aldrich) with gentle vortexing. The gel spots were then washed with double distilled water and equilibrated with 100 mM Ammonium Bicarbonate (Cat. No. A6141, Sigma). Gel spots were then dehydrated in Acetonitrile (Cat. No. 34967, Riedel-de Haën). DTT and IAA were added respectively into dehydrated gel spots. The gel spots were then dehydrated again with acetonitrile before 10 ng/ μ! Trypsin (Cat. No. V5280, Promega) was added for digestion overnight at 37°C. Peptides were extracted using 50% ACN/5% Trifluoroacetic Acid (TFA). The peptides were then dried using vacuum dry method and cleaned with ZipTip® C18 (Cat. No. ZTC 18S 096, Millipore) according to manufacturer’s instruction.
Mouse genomic DNA extraction
Mouse tail was cut and digested in 495 μl NTES buffer (50 mM Tris-Cl, 50 mM EDTA, 100 mM NaCl, 5 mM DTT, 0.5 mM spermidine and 2% SDS) and 5 μl of proteinase K (Cat. No. 13215100, Roche) overnight at 55°C in a rotary oven. The next day, equilibrated phenol (Cat. No. C2432, Sigma), phenol:chloroform:isomyl alcohol (Cat. No. 75831, USB) and chloroform (Cat. No. 75829, USB) were sequentially used for purification of protein from DNA extract. The genomic DNA was then precipitated by isopropyl alcohol (Cat. No. A415-4, Fisher) and dissolved in TE buffer.
Mouse genotyping
Three primers named oIMR1965, oIMR1966 and oIMR1967 were used to determine the genotype of the mouse. Primer oIMR1965 was the common primer; when it paired with primer oIMR1966, a 700 bp fragment from the wild type allele would be amplified. On the other hand, when primer oIMR1965 paired with primer oIMR1967, a 320 bp fragment from mutant allele would be amplified. The PCR cycling condition was heat activation at 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 67°C for 1 min and 72°C for 1 min; the final extension step was done at 72°C for 2 min. The PCR product was analyzed by electrophoresis on 1.5% agarose gel.
Real time RT-PCR
The reaction was performed using iTaqTM SYBR® Green Supermix with ROX (Cat. No. 1725850, Bio-Rad) containing 2X reaction buffer, 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, 0.8 mM dUTP, iTaq DNA polymerase, 6 mM Mg+2, SYBR Green I dye, 1 μM ROX internal reference dye and stabilizers. Reaction volume used was 25 μ, including 12.5 μl of 2X SYBR® Green Supermix, 1 μl of synthesized cDNA, 1 μl of 10 μM forward primer, 1 μl of 10 μM reverse primer [Table 2], topping up with nuclease free water.
Cycling was performed on 7500 Real Time RT-PCR system (Applied Biosystem). Cycling conditions were machine warm up at 50°C for 2 min, hot initiation at 95°C for 10 min, cycling condition (45 cycles) of 95°C for 30 s, 60°C for 30 s and 72°C for 2 min, followed by a dissociation stage to generate a melting curve. AACT method was employed to calculate the differential expression of mRNA in samples examined, by comparing cycling results between target gene and basal control Glyceraldehyde-3-phosphate dehydrogenase (GADPH).
|
Name |
Sequence |
|
HSP70 |
Sense: 5′-AAG AAC GCG CTC GAG TCC TAT GC-3′ |
|
Anti-sense: 5′-CAC CCT GGT ACA GCC CAC TGA TGA T-3′ |
|
|
CaBP |
Sense: 5′-GAT GGC AAC GGA TAC ATA GAT GAA-3′ |
|
Anti-sense: 5′-TCC ATC CGA CAA GGC CAT TAT GTT C-3′ |
|
|
GADPH |
Sense: 5′-AGT CTA CTG GTG TCT TCA CCA CCA TGG-3′ |
|
Anti-sense: 5′-TTC TCG TGG TTA ACA CCC ATC AC-3′ |
|
|
VDR |
Sense: 5′-AGG TGC AGC GTA AGC GAG AGA T-3′ |
|
Anti-sense: 5′-CCT CAA TGG CAC TTG ACT TAA GC-3′ |
|
|
NeuroD |
Sense: 5′-CTC AGT TCT CAG GAC GAG GA-3′ |
|
Anti-sense: 5′-TAG TTC TTG GCC AAG CGC AG-3′ |
|
|
Pax6 |
Sense: 5′-AGT CAC AGC GGA GTG AAT CAG-3′ |
|
Anti-sense: 5′-AGC CAG GTT GCG AAG AAC-3′ |
|
|
Mash1 |
Sense: 5′-AGC AGC TGC GGA CGA GCA-3′ |
|
Anti-sense: 5′-CCT GCT TCC AAA GTC CAT TC-3′ |
|
|
LDH |
Sense: 5′-AGC AAA GAC TAC TGT GTA ACT GCG A-3′ |
|
Anti-sense: 5′-ACC TCG TAG GCA CTG TCC AC-3′ |
|
|
MDH |
Sense: 5′-AGG CTA CCT TGG ACC GGA GCA GTT-3′ |
|
Anti-sense: 5′-GTG GCA GAA CCT GCT CCA GCC TT-3′ |
|
|
Glutathione S- |
Sense: 5′-TGA CGC TCC CGA CTT TGA CAG AA-3′ |
|
Transferase Mu1 |
Anti-sense: 5′-TAA GCA AGG AAA TCC ACA TAG GTG-3′ |
|
ATP synthase 5a1 |
Sense: 5′-AGA AGA CTG GCA CAG CTG AGA TGT-3′ |
|
Anti-sense: 5′-CCA GTC TGT CTG TCA CCA AT-3′ |
|
|
SOD2 |
Sense: 5′-ATG AAA GCC ATC TGC ATC ATT AGC-3′ |
|
Anti-sense: 5′-GCA ATT ATT CCG CAT CCC AAA CG-3′ |
|
|
NSF |
Sense: 5′- TGG GGC AGC AGC TTG TCT TTA -3′ |
|
Anti-sense: 5′- TTA GCA CCA AGC CTC CTT TGC -3′ |
Table 2. Primer sequences used in Real Time RT-PCR analysis:
Western blot analysis
Protein homogenates were heated in Laemmli sample buffer (Cat. No. 161-0737, Bio-Rad), at 95°C for 10 min. The heated samples were resolved on 12% SDS-PAGE gel and transferred to Immun-Blot PVDF membrane (Cat. No. 162-0177, Bio-Rad) at 100 V for at least 1 hr. The membrane was then blocked with 5% non-fat milk in 0.1% T-TBS (10 mM Tris-Cl, pH 7.5, 150 mM NaCl and 0.1% Tween-20; NaCl Cat. No. 1.06404, Merck, Tween-20 Cat. No. 1610781, Bio-Rad ) for 1 hr at RT. The membrane was then washed three times in 0.1% T-TBS, followed by incubation with primary antibody [Table 3] for 1 hr. The membrane was then washed again three times in 0.1% T-TBS and incubated for 1 hr with HRP conjugated secondary antibody. The membrane was then washed again three times in 0.1% T-TBS before being developed by ECL method (Cat. No. RPN 2108, ECL Western blotting analysis system, Amersham). The blot was stripped with 0.1% T-TBS overnight on an orbital shaker for second antibody detection. The intensity of protein bands detected by Western blot analysis was determined by calibrated densitometer GS-800 and Quantity One 1-D analysis software (Cat. No. 170-7983, Bio-Rad).
|
Antibody name |
Dilution factors used in experiment |
|
Anti-E6AP (Cat. No. A300-352A, Bethyl) |
1:2500-5000 |
|
Anti Calbindin D-28k (Cat. No. AB1778, Chemicon) |
1:2500-5000 |
|
Anti-Actin (Cat. No. MAB1501, Chemicon) |
1:1000-2500 |
|
Secondary HRP conjugated anti-mouse/anti-rabbit (GE Healthcare) |
1:2500-5000 |
|
Anti-Hsp70 (Cat. No. sc32239 , Santa Cruz) |
1:2500-5000 |
|
Anti-SOD2 (Cat. No. sc-30080, Santa Cruz) |
1:2500-5000 |
|
Anti-NSF (Cat. No. ab16681, abcam) |
1:10000 |
|
Anti-Mash1 (Cat. No. AB155582, Chemicon) |
1:2500-5000 |
|
Anti-NeuroD (Cat. No. AB155580, Chemicon) |
1:2500-5000 |
Table 3. Antibodies used in experiments
