Additional file 1 of Mouse models characterize GNAO1 encephalopathy as a neurodevelopmental disorder leading to motor anomalies: from a severe G203R to a milder C215Y mutation
Creators: name: Silachev, Denis
name: Koval, Alexey
name: Savitsky, Mikhail
name: Padmasola, Guru
name: Quairiaux, Charles
name: Thorel, Fabrizio
name: Katanaev, Vladimir L.
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Neuroscience
subject: Developmental Biology
subject: Cancer
subject: 111714 Mental Health
subject: FOS: Health sciences

description: Additional file 1: Fig. S1. Transcription of G203R and C215Y alleles. (A) Schematic exon-intron structures of GNAO1. Black and white boxes mark the coding and noncoding parts of the exons respectively. The drawing of the exons is presented in scale. Digits inside the exons indicate their precise length in base pairs. Red arrows flag mutations in the 6th exon. Primers used in RT-PCR are designated with black horizontal arrows. (B) Agarose gels with RT-PCR products obtained for different genotypes with the primers 1 and 2 as shown on panel (A). As expected, all resultant products migrate in gels as 160bp bands. (C) Sequences of RT-PCR products for GNAO1 mutants and the wt (+/+) control. Codons, amino acids with their numbers are shown above the sequence of the wt. Black dashed vertical line demarcates the 5th and 6th exons. Red dashed vertical lines designate codons for the 203rd and 215th amino acids of Gαo. Note that the peak height of the mutant nucleotide is nearly identical to that of wild-type in both mutants, speaking of near-equal expression of both alleles. Fig. S2. Normal striatum morphology in C215Y mutants. (A) Staining of brain sections with striatum identified with the Ctip2 marker (marks GABAergic medium-sized spiny neurons) from wt (+/+), C215Y/+, and C215Y/C215Y E18.5 embryos. (B) DAPI staining of the same sections. Note the enlarged lateral ventricles (right to the striatum) in the C215Y mutants. (C) Quantification of Ctip2-positive cells. (D) Quantification of the cell density by DAPI-stained cells in striatum. Data is presented as mean ± SD; n = 3 animals. Scale bar: 0.5mm. Fig. S3. Normal hippocampus morphology in C215Y mutants. (A) Cresyl violet staining of coronal brain sections from wt (+/+), C215Y/+, and C215Y/C215Y E18.5 embryos does not reveal significant changes in the morphology of hippocampus. Scale bar: 100μm. (B) Magnified photomicrographs of CA1 subfield of the hippocampus, marked with a rectangle in (A). Scale bar: 20μm. (C) The graph summarizing CA1 neuronal counts as Data is presented as mean ± SD; n = 3 animals. Fig. S4. Analysis of the upper layer cortical neurons and Cajal-Retzius cells in C215Y mutants. Staining of motor cortex brain sections from E18.5 wt (+/+), C215Y/+, and C215Y/C215Y mice for DAPI and Brn2 (layers II-V (A)), or DAPI and Reelin (marker of Cajal-Retzius cells, (B)). (C) Quantification of Brn2-positive cells. (D) Quantification of Reelin-positive cells. Data is presented as mean ± SD; n= 3-5 animals; **p&lt;0.01, significance from the wt is assessed by t-test. Scale bar: 20μm (A) and 50μm (B). Fig. S5. Normal levels of apoptosis in C215Y brain sections. The distribution of cleaved caspase-3 (CC3)-positive cell in the brain sections from E18.5 wt (+/+), C215Y/+, and C215Y/C215Y mouse embryos. (A) Representative immunohistochemistry pictures of cleaved caspase-3 (CC3)-positive cells (marked with arrows) in the cortical plate, scale bar 20μm. The counting of caspase-3 positive cell in the cortical plate (B), the subventricular zone (C) and the ventricular zone (D). Data is presented as mean ± SD, n= 3; t-test did not reveal any statistically significant differences from wt. Fig. S6. G203R mutation affects brain morphology, neurons in deep and upper cortical layers as well as neural progenitor cells. (A) Hematoxylin and eosin staining of coronal brain sections from G203R/+, G203R/G203R and wt (+/+) littermates at embryonic day 18.5 shows enlargement of lateral ventricles in G203R/+ and reduction in G203R/G203R mice. (B) Coronal sections through the motor cortex show differences in cortical thickness between wt and G203R/+ mice. (C) Staining of brain sections (at motor cortex) from E18.5 G203R/+, G203R/G203R and +/+ littermates for Tbr1 (layer VI), Ctip2 (weak in layer VI, strong in layer V), Brn2 (layer II/III), Reelin (marker of Cajal-Retzius cells), Tbr2 (marker of intermediate neuronal progenitor cells) and Nestin (marker of radial glial cells). Scale bars: 1mm (A), 100µm (B, C). Fig. S7. Additional data for the open field and pole tests. (A) Open field test: group mean of the time in the center of open field arena for the +/+, C215Y/+, and C215Y/C215Y mice. (B) C215Y/+ mice remained more mobile than wt in the open field test despite the overall suppression of motility induced by risperidone treatment (0.1mg/kg). Data is shown as distance moved by a mouse in the period of 30min of the experiment. (C) Pole test: means of time to a T-turn and time to descend in the pole test for the three genotypes. Data are presented as in Figure 5. Fig. S8. MoSeq quantification of the exploratory behavior under unchallenged conditions. The modified MoSeq algorithm was applied to monitor and analyze behavior of C215Y/+ and C215Y/C215Y female mice, alongside with their wt littermates, under non-challenged conditions in a square open field. The heatmaps demonstrate comparisons among the three genotypes for the syllable (behAdditional file 1: avioral pattern) frequency (number of times this syllable starts per minute of activity), mean length (the time the syllable lasts before changing to another syllable) and its overall proportion in percent over the entire analyzed time. Statistical significance was assessed by Dunn's multiple comparison test for each syllable and is shown as a number of compared group on top of the heatmap cell in case of a significant difference (p value &lt;0.05), numbers are shown side by side in case of significant differences in 2 groups. Syllable description is provided below the heatmaps. Fig. S9. Detailed view of selected syllables representing majority of forward, sitting and rearing-related movements. The main characteristics of these syllables shown in Fig. S9 as a heatmap represented here as bar charts. Significance was assessed as in Fig. S7, *p&lt;0.05 , **p&lt;0.01, ***p&lt;0.001. Fig. S10. Networks analysis of MoSeq behavioral data. Transitions between behavioral syllables for wt (A), C215Y/+ (B), and C215Y/C215Y (C) animals. The syllables are shown as nodes and grouped by their gross appearance. The node size corresponds to the overall proportion of the syllable over the recording time (as shown on heatmaps in Fig. S8), nodes of syllables showing significant differences in at least one aspect (frequency, length or proportion,) are marked in red. Edge thickness corresponds to a proportion (in %) of a given transition pair among all recorded transitions for the genotype, purple numbers are shown for edges with thickness &gt; 0.2. Edges with thickness &lt;0.01 represent rare transitions and are thus not shown. Fig. S11. Absence of epileptic manifestations in C215Y/+ mice. Surface EEG recordings in control and mutant awake mice. (A) The head-fix epicranial EEG recording setup. The right image illustrates the localizations of the electrodes over the mouse brain. In red, the electrodes positioned above the somatosensory cortex regions. (B) Examples of 10sec windows of background EEG recorded in both control and mutant mice. (C) Illustrative examples of physiologic fast ripples (FRs) recorded over the somatosensory cortex in control and mutant mice as visible in the raw signals and in the signal filtered between 200-550Hz. (D, E) Pentylenetetrazole (PTZ)-induced mouse kindling reveals no difference between C215Y/+ and wt mice. Scheme of the experiment is shown in (D). In (E), seizure score quantification at three different injections of PTZ. Data are shown as mean ± SD, n=5 (wt) and 7 (C215Y/+) mice.