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Supplementary Information
Table S1
Metabolomics analysis of Asc-1-KO mice (P7).
Table S2
Comparison of Asc-1-KO with mice models of hyperekplexia.
Movie S1
Impaired righting response and handling-induced tremors in Asc-1-KO mice (P15).
Movie S2
Effect of glycine (28 mmol/Kg i.p.), D-serine (9 mmol/Kg i.p.) and L-serine (28
mmol/Kg i.p.) administration on the motor phenotype of P19 (glycine and D-serine) and
P14 Asc-1-KO mice (L-serine).
Fig. S1 - Hind-feet clasping reversal by glycine is preventable by strychnine.
Asc-1-KO mice (P14) were injected with glycine (28 mmol/Kg i.p.) and hind-feet
posture was monitored after an hour. Mouse on the right receive an injection of
strychnine (1 mg/Kg, i.p.) 10 minutes prior to glycine injection.
Fig. S2 - Effects of glycine, L- and D-serine on amino acid levels in Asc-1-KO mice
brains.
A. Glycine levels in the cerebral cortex (Ctx) of Asc-1-KO mice upon saline, glycine (28
mmol/Kg i.p.), L-serine (28 mmol/Kg i.p.) or D-serine injection (9 mmol/Kg i.p.).
Amino acids were analyzed 3 h (saline, glycine and D-serine) or 2 h (L-serine) after
administration.
B. Glycine levels in the pons-medulla (P-M) of Asc-1-KO mice following the
treatments described in (A).
C. L-Serine levels in the Ctx of Asc-1-KO mice after the treatments described in (A).
D. L-Serine levels in the P-M of Asc-1-KO mice after the treatments described in (A).
E. GABA levels in the Ctx of Asc-1-KO mice after the treatments described in (A).
F. GABA levels in the P-M of Asc-1-KO mice after the treatments described in (A).
G. Glutamate levels in the Ctx of Asc-1-KO mice after the treatments described in (A).
H. Glutamate levels in the P-M of Asc-1-KO mice after the treatments described in (A).
I.
Levels of D-serine in the cortex.
J. Same as in (I), but in the pons-medulla (P-M).
The results are average ± SEM of 4 (Glycine and L-serine injected) or 3 (D-serine
injected) Asc-1-KO mice at P17-20. *,**,*** Different from WT at p<0.05, 0.01 and
0.001, respectively (One-way ANOVA and Bonferroni's post-hoc test).
Fig. S3 - Expression of glycinergic transmission components and metabolic enzymes
in Asc-1-KO mice.
A. Western-blot analysis of glycine receptor 1 (GLRA1) and  (GLRB), GlyT2, GlyT1,
VGAT, gephyrin and -tubulin expression in the cortex (Ctx) and pons-medulla (P-M)
of WT and Asc-1-KO mice (P14).
B. Western-blot of GCSH, AMT, SHMT1, SHMT2, Phgdh and -tubulin expression in
the Ctx and P-M of WT and Asc-1-KO mice (P14).
C. Asc-1-KO/WT expression ratio in the Ctx.
D. Asc-1-KO/WT expression ratio in the P-M.
AMT; aminomethyltransferase; GCSH, glycine cleavage system H; GlyT1,
glycine transporter 1; GlyT2, glycine transporter 2; Phgdh, phosphoglycerate
dehydrogenase; SHMT1, serine hydroxymethyl transferase 1; SHMT2,
serine
hydroxymethyl transferase 2; VGAT, vesicular GABA (and glycine) transporter.
The values in C and D are the average ± SEM of the Asc-1-KO/WT expression
ratio of independent Western-blots from 3 (AMT, GCSH, Phgdh, SHMT1), 4 (GlyT1,
SHMT2) or 5 (GLRA1, GLRB, GlyT2) WT and Asc-1-KO mice at P14 and 20. The
values were obtained by quantification of the luminescence signal and normalization by
the -tubulin expression in each individual blot. Student's t test showed no difference in
the expression between the groups.
Fig. S4 - Effect of D-Ala on L-serine uptake by pons-medulla synaptosomes.
Effect of D-Ala (1 mM) on the uptake of L-[3H]serine by pons-medulla synaptosomes
from WT when compared to Asc-1-KOs. The data are the average ± SEM of 4
independent experiments with different synaptosomal preparations from P14-18 mice.
***, Significant difference from control lacking D-Ala at p<0.001 (Paired Student's t
test).
Supplementary Methods
Startle reflex and tremor evaluation- Each session start with 3 minutes acclimatization
period in a constant 57 dB background white noise level and it is delivered continuously
throughout the test session (Kinder Scientific, CA). To evaluate the startle response, mice
were exposed to three startle intensities (four trials each intensity, 12 trials total) at 40 ms
duration pulse (100, 110 and 120 dB) in a random inter-trial-interval (range between 1020 sec, average of 15 sec) as well as in a pseudo-random order, in order to avoid
habituation. In order to evaluate spontaneous tremors we employed additional 8 pseudorandom recording windows/ trials order without the auditory stimuli.
Open Field- The open field was made from black lusterless acrylic box (50L × 50W × 40H
cm) placed in a dimly lit room (50 lux). The floor was scattered with sawdust bedding in
order to minimize anxiety and body temperature loss. Mice were tested for activity
between the ages of 17-19 days, placed in the corner of the open field (facing the wall)
and given 5 min of free exploration. The behavior was videotaped by a color CCTV
Panasonic camera (WV-CP500(. Activity was post-recording analyzed using Ethovision
XT 9.0 software (Noldus, The Netherlands), as described previously [1].
Statistical analysis of behavioral tests- Data were analyzed using a Two-way ANOVA for
mixed design with group as between subject factor and test-retest as within subject factor.
Post-hoc student’s t test was calculated between the two groups (SPSS v.21). A result
was considered significant when P-value was less than 0.05. Results are displayed as
mean± S.E.M.
Western-blots- Cerebral cortici and pons-medulla were dissected and homogenized with
20 strokes of a glass homogenizer in 10 volumes of buffer consisting of 20 mM Tris-HCl
(pH 7.4), 50 mM NaCl and protease inhibitors (Mini-complete, Roche). Samples were
cleared by centrifugation twice at 1500 x g at 4oC and 40 g of supernatants were
analyzed by SDS-Page and western-blot. Expression levels were quantified by
monitoring the densitometry of the chemiluminescent signal in each lane (Image Quant,
GE Healthcare) and normalized by their respective -tubulin levels. The blots were
assembled with Adobe Photoshop (Version C52) without changing contrast or brightness
parameters. The following antibodies were used for Western-Blots:
Antigens
Company
Catalog
Dilution
GLRA1
Synaptic Systems
146003
1:1000
GLRB
Santa Cruz
sc-17285
1:1000
GlyT2
Millipore
AB1773
1:10000
GlyT1
Millipore
AB1770
1:3000
VGAT
Millipore
AB2257
1:1000
Gephyrin
Millipore
AB5725
1:200
GCSH
Sigma-Aldrich
HPA041368
1:1000
AMT
Santa Cruz Biotechnology sc-99267
1:1000
SHMT1
Cell Signaling
12612
1:1000
SHMT2
Cell Signaling
12762
1:1000
Phgdh
Frontier Biosciences
Af303
1:1000
β -Tubulin Sigma-Aldrich
Clone SAP 4G5 1:1000
Synaptosomal preparation- Cerebral cortici or pons-medulla were dissected on ice and
homogenized in 20 volumes of 0.32 M sucrose and 1 mM EDTA with 10 strokes of a
Teflon homogenizer at 800 rpm. The homogenate was centrifuged at 1000 x g for 5 min
to remove nuclei and debris and the resulting fraction (S1) was layered onto a
discontinuous Percoll gradient. Purified synaptosomes were prepared by collecting the
10-23% interface as previously described [24]. The synaptosomal fraction was diluted in
0.32 M sucrose, 1 mM EDTA and 20 mM Hepes-NaOH and washed by 30 min
centrifugation at 30,000 x g. Then, the synaptosomal pellet was resuspended at a
concentration of about 1 mg protein/ml in HBSS solution, with the following
composition (mM): 137 NaCl, 5.4 KCl, 0.95 CaCl2, 0.44 KH2PO4, 0.406 MgSO4, 0.492
MgCl2, 4.2 NaHCO3, 0.34 Na2HPO4 x12, 1 Na pyruvate10 HEPES, pH 7.4, 5.6 glucose,
pH 7.4. The preparations were stored on ice and used within 30 minutes for amino acid
uptake.
Materials- L-serine was purchased from Bachem. Boc-L-cysteine, dithiothreitol (DTT),
glycine, o-phtalodialdehyde, percoll, proteinase K, D-serine were purchased from SigmaAldrich. Milacemide (Lot B13Z1127) was custom synthesized by Boc Sciences. L-[U14
C]-serine (Lot 1792180), glycine [2-3H] (Lot 1817306 and 3632835 were obtained from
Perkin Elmer. L-serine[3-3H] (Lot 131017) was purchased from American Radiolabeled
Compounds.
References
1-Spink AJ, Tegelenbosch, RAJ, Buma, MOS, Noldus, LPJJ, (2001) The EthoVision
video tracking system- A tool for behavioral phenotyping of transgenic mice. Physiol. &
Behavior. 73: 731– 744.