Evaluation of the effects of the T-type calcium channel enhancer SAK3 in a rat model of TAF1 deficiency

The TATA-box binding protein associated factor 1 (TAF1) is part of the TFIID complex that plays a key role during the initiation of transcription. Variants of TAF1 are associated with neurodevelopmental disorders. Previously, we found that CRISPR/Cas9 based editing of the TAF1 gene disrupts the morphology of the cerebral cortex and blunts the expression as well as the function of the CaV3.1 (T-type) voltage gated calcium channel. Here, we tested the efficacy of SAK3 (ethyl 8′-methyl-2′, 4-dioxo-2-(piperidin-1-yl)-2′H-spiro [cyclopentane-1, 3′-imidazo [1, 2-a] pyridine]-2-ene-3-carboxylate), a T-type calcium channel enhancer, in an animal model of TAF1 intellectual disability (ID) syndrome. At post-natal day 3, rat pups were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 CRISPR/Cas9 viruses. At post-natal day 21, the rat pups were given SAK3 (0.25 mg/kg, p.o.) or vehicle for 14 days (i.e. till post-natal day 35) and then subjected to behavioral, morphological, and molecular studies. Oral administration of SAK3 (0.25 mg/kg, p.o.) significantly rescued locomotion abnormalities associated with TAF1 gene editing. SAK3 treatment prevented the loss of cortical neurons and GFAP-positive astrocytes observed after TAF1 gene editing. In addition, SAK3 protected cells from apoptosis. SAK3 also restored the Brain-derived neurotrophic factor/protein kinase B/Glycogen Synthase Kinase 3 Beta (BDNF/AKT/GSK3β) signaling axis in TAF1 edited animals. Finally, SAK3 normalized the levels of three GSK3β substrates - CaV3.1, FOXP2, and CRMP2. We conclude that the T-type calcium channel enhancer SAK3 is beneficial against the deleterious effects of TAF1 gene-editing, in part, by stimulating the BDNF/AKT/GSK3β signaling pathway.

Together, SAK3-mediated rescue of cognitive impairment, likely via its activation of CaV3.1, make this small molecule an attractive drug candidate for ameliorating symptoms of TAF1 ID syndrome.
We have previously shown that TAF1 gene editing alters the morphology of the cerebral cortex (Janakiraman et al., 2019). Our group and others have also shown that TAF1 gene editing decreases the expression and function of the CaV3.1 T-type calcium channel in vitro and in vivo (Hurst et al., 2018;Gudmundsson et al., 2019;Janakiraman et al., 2019). We also showed that SAK3 treatment reverses the loss of purkinje cells after TAF1 gene editing. (Janakiraman et al., 2020). Therefore, here we investigated the potential beneficial effects of SAK3 treatment on the cerebral cortex. Within each cortical layer, there are several subtypes of pyramidal neurons. We chose to analyze somatosensory motor cortex pyramidal layer 5 for analysis because of their large soma size and broad-tufted cells of the motor cortex (Jiang et al., 2020). We also provide new insights into the signaling of SAK3 in the central nervous system.

Animals
Pathogen-free, normal E18 pregnant Sprague-Dawley rats (Envigo Laboratories) were housed 1 per cage in temperature-(23 ± 3 °C) and light (12-h light/12-h dark cycle; lights on 07:00-19:00)-controlled rooms with standard rodent chow and water available ad libitum. The neonates are designated as post-natal day 0 (PD0) on the day of birth, the litter size range included in the current study was 11-14 pups.
After weaning the rat pups were separated from the dams and maintained 4 per cage. Animals were divided into six groups. SAK3 (Catalog No: SML-2039-5MG, Sigma Aldrich) was dissolved in distilled water and orally administered (0.25 mg/kg, p.o) to the animals from postnatal day 21 to 35 (PD21-35) (Fig. 1A). We chose to administer this dose of SAK3 because it has been shown to be effective in restoring cognitive function in a mouse model of Alzheimer's disease . Rats were sacrificed at PD35. Animals were behaviorally assessed before being euthanized for histological, immunostaining and protein expression analysis. All biochemical and behavior experiments were performed in a blinded fashion. Animal protocols were approved by the Institutional Animal Care and Use Committee of the College of Medicine at the University of Arizona and conducted in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health.

CRISPR/Cas9-mediated targeting of TAF1 gene
Our strategy to truncate TAF1 focused on targeting exon 1 of the TAF1 gene using a guide RNA (gRNA) has been described previously (Moutal et al., 2017;Moutal et al., 2018a,b;Sandweiss et al., 2018). We targeted this exon to ensure total removal of the TAF1 protein.
Using this approach, we expect minimal to none off-target activity of the Cas9 enzyme as we and others verified before (Moutal et al., 2017;Ma et al., 2017). The gRNA sequence (GTGTCTGACATGACGGCGGA, quality score 94) was inserted into the restriction site of the pL-CRISPR.EFS. tRFP lenti-plasmid (Cat#57819, Addgene, Cambridge, MA) (Heckl et al., 2014), a plasmid that allows for simultaneous expression of (i) the Cas9 enzyme; (ii) the gRNA; and (iii) a red fluorescent protein (tRFP) -to control for transduction efficiency. All plasmids were verified by Sanger sequencing (Eurofins, Louisville, KY). Lenti-viral plasmids were packaged in lentiviruses by Viracore (USCF, CA) at titers routinely above 10 7 infectious particles per ml.

Intracerebroventricular injections
Bilateral intracerebroventricular (ICV) injections were performed as previously described in Sprague-Dawley (SD) rat pups on postnatal day 3 (Delenclos et al., 2017;Pang et al., 2003). Briefly, newborn SD rat puts were anesthetized by isoflurane. A 10 μl syringe (Hamilton Gastight Syringe, #1701) was used to pierce the skull (coordinates from bregma: − 0.6 mm posterior, ± 1.75 mm lateral/medial, and − 2.5 mm ventral), and 2.5 μl of CRISPR/Cas9 lentivirus (gRNA-control or gRNA-TAF1) was injected into each cerebral ventricle. Previous studies demonstrated that this route of delivery allows for efficient reduction of TAF1 expression in all cells of the cerebellum, cerebral cortex and hippocampus (Janakiraman et al., 2019). Neonatal rat pups were kept with the dam until weaned.

Open field test
Open field test is used for analyzing the behavior and locomotion in rats. The apparatus is 120-cm diameter circular arena, bordered by a 50-cm-high wall made of wood. The floor of this chamber was divided into central and peripheral zone. The rat pups were placed into the peripheral zone of an open field chamber and locomotion was observed for 5 min (Rajasankar et al., 2009).

Nissl staining
After dehydration of the tissue with 30% sucrose, 20 μm sections were cut, stained with cresyl violet dye (Catalog No: C5042-10G, Sigma), and mounted with Richard-Allan scientific mounting medium (Catalog No: 4112, Thermo Scientific) then visualized the somatosensory motor cortex pyramidal layer 5 of motor cortex.

Cell counting
Twelve visual fields (0.6 mm2) of the somatosensory motor cortex pyramidal layer 5 of the motor cortex were randomly imaged from each section. The number of stained cells in each field was counted at a 40× magnification. Data were expressed as the number of cells per field (Liu et al., 2011).

TUNEL assay
Cerebral cortex tissue sections of 20 μm were cut and 5-7 sections chosen according to systematic random sampling scheme from each sample were processed with In Situ Cell Death Detection Kit, Fluorescein (Catalog No: 11684795910; Roche, Millipore Sigma, USA) according to the manufacturers protocol for tissues. Then we focused on the somatosensory motor cortex pyramidal layer 5 of the motor cortex using a 20× objective and apotome 2(LSM510, Carl Zeiss).

Immunohistochemistry
The antibody used in our studies are listed in Table 1. Rats (n = 6 animals per experimental condition) were perfused with saline and 4% formaldehyde in Phosphate buffer saline (PBS) at PD35, and the brains were extracted and post-fixed in 4% paraformaldehyde for 8 h at 4 °C. Cerebral cortex sections were cut sagittal at 20 μm using a cryostat (Microm HM 505 E). After rinsing the sections in PBS for 5 min, the sections were incubated with a 0.1% H2O2 solution in PBS for 5 min, rinsed in PBS for 5 times for 5 min, and incubated for 30 min with 0.4% Triton X-100, rinsed in PBS and blocked with 8% goat serum, and 1% Triton X-100 in PBS. After blocking, the sections were incubated at 4 °C for overnight with the indicated antibodies diluted in 4% goat serum in PBS. The sections were washed in 1% goat serum in PBS, incubated with secondary antibody anti-rabbit Alexa fluor 488 (Life Technologies) or anti-mouse Alexa fluor 488 (Life Technologies), as needed, in 4% goat serum for 2 h, washed with PBS 3 times for 5 mins, and incubated with DAPI (Catalog No: D1306, Thermofisher Scientific) at concentration of 50 ng/ml for 2 mins. Sections were then washed and further air dried, and cover slipped with glycerol. All procedures were performed at room temperature. Stained slides were observed under a fluorescence microscope, focusing on the somatosensory motor cortex pyramidal layer 5 using a 20× objective and apotome 2 (LSM510, Carl Zeiss). Immunflourescence was quantified in 12 different fields from 4 different animals per experimental as previously described (Janakiraman et al., 2019).

Statistics
All data was first tested for a Gaussian distribution using a D'Agostino-Pearson test (Graphpad Prism 8 Software, Graphpad, San Diego, CA). The statistical significance of differences between means was determined using a parametric ANOVA followed by Tukey's post-hoc test or a non-parametric Kruskal Wallis test followed by Dunn's post-hoc test depending on if datasets achieved normality. Differences were considered significant if p ≤ 0.05. Error bars in the graphs represent mean ± SEM.

SAK3 treatment improves locomotion
We used the Open Field Test to measure locomotor activity and anxiety-like behavior in the animals. We found that TAF1 edited animals displayed decreased locomotion compared to the controls and SAK3 treatment increased locomotion of TAF1 edited animals (Fig. 1B). We also measured thigmotaxis, a parameter of anxiogenic behavior in rodents (Seibenhener and Wooten, 2015). We found no difference in thigmotaxis between the experimental groups ( Fig. 1C&D).

SAK3 treatment attenuates the morphological abnormalities caused by TAF1 deletion
We previously documented that TAF1-editing caused morphological abnormalities in the cells of the cerebral cortex (Janakiraman et al., 2019), so we evaluated the morphology of the neurons in layer 5 of the motor cortex by Nissl staining. Our histopathology analysis showed that neurons in the control group were arranged tightly with regular pyramidal morphology at layer 5 within the sensory-motor cortex and had an intact cell structure. The blue Nissl bodies in the neurons were visible and distinct. As we previously reported, the morphology of the neurons in the motor cortex at layer 5 was clearly different in TAF1edited animals. The shape of the neurons changed from a pyramidal to a stellate morphology and many neurons appeared to be vacuolated with enlarged intercellular spaces. Also, the neuronal cells did not stain as distinctly as control neuronal cells with the Nissl dye ( Fig.  2A). However, SAK3 administration improved the morphology of the neurons in cortex ( Fig. 2A). In TAF1-edited animals, we found a drastically reduced number of neurons compared to Naïve or gRNA-control rats (Fig. 2B). SAK3 administration resulted in an increased number of neurons compared to TAF1-edited animals (Fig. 2B). Treatment with SAK3 had no effect on the Naïve and gRNA-control group compared to the vehicle (water) (Fig. 2B). Collectively, these results strongly suggest that TAF1 is directly implicated in neuronal survival.
Among the essential cell types in the brain, astrocytes are the most abundant population. Astrocytes retain proliferative capacity, and their functions are crucial for neuronal survival (Jakel and Dimou, 2017). Astrocytes are critical for mediating ion homeostasis, growth factor responses and neurotransmitter functions in the brain (Devinsky et al., 2013). We previously reported a decrease in GFAP-positive astrocytes within the granular layer of the cerebellum in TAF1-edited animals (Janakiraman et al., 2019). Similarly, we found decreased numbers of astrocytes in the cerebral cortex of TAF1-edited rats treated with SAK3 (Fig. 2C, D). Thus, morphological alterations of neurons and astrocytes induced by TAF1 editing in layer 5 of the cerebral cortex can be reversed by SAK3 treatment.

Administration of SAK3 attenuates apoptosis
Because we observed morphological abnormalities and decreased cell viability in TAF1 edited animals which was rescued by SAK3, we assessed if apoptosis could be enhanced in TAF1 edited animals. We observed an increase in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells in layer 5 of the cerebral cortex in TAF1 edited animals. SAK3 treatment reduced the number of TUNEL positive cells to control levels in TAF1 edited animals (Fig. 3A, B). In agreement with our previous observations (Janakiraman et al., 2020), no significant changes in the TUNEL positive cells of water and SAK3 treatment to the naïve and gRNA-control animals were observed. The protective effects of SAK3 against apoptosis was corroborated by (i) the down-regulation of activated caspase 3 (Fig. 4A, B); (ii) the down-regulation of the pro-apoptotic factor BCL2 Associated X (BAX), an apoptosis regulator (Fig. 4C, D); and (iii) up-regulation of B-cell lymphoma 2 (Bcl-2), an apoptosis suppressor gene (Fig. 4E, F).

SAK3 treatment stimulates Brain-derived neurotrophic factor/protein kinase B/ Glycogen Synthase Kinase 3 Beta (BDNF/AKT/GSK3β) signaling in the cerebral cortex of TAF-1 edited animals
We examined the BDNF/AKT signaling pathway because previous studies demonstrated that BDNF can protect cortical neurons from apoptosis (Poser et al., 2003). Consequently, we investigated whether SAK3 treatment could be neuroprotective through this pathway. We observed that BDNF levels were decreased in TAF1 edited animals compared to naïve animals or injected with gRNA-control CRISPR. SAK3 administration restored BDNF expression to the levels seen in naïve and gRNA-control groups (Fig. 5A-D). Protein kinase B (AKT) phosphorylation at Ser 473 is associated with its activation (Aneichyk et al., 2018). We found that activated AKT (phosphorylated at Ser 473) was decreased in the cortex of TAF-1 edited animals which was prevented by SAK3 treatment (Fig. 6A, B). Glycogen synthase kinase 3 (GSK3) has two isoforms, GSK3α and GSK3β, that are inactivated by AKT phosphorylation on Ser 21 and Ser 9, respectively (Cross et al., 1995). Inhibition of GSK3β is known to protect against apoptosis in many situations (Pap and Cooper, 1998). GSK3β is a downstream target of AKT and activation of AKT inhibits GSK3β by inducing its phosphorylation (Wu et al., 2007). We found that phospho (Ser 9)-GSK3β levels were decreased in TAF1 edited animals and SAK3 treatment increased phopho-GSK3β levels (Fig. 6C, D). Similar effects were seen in the cerebellum (Suppl. Fig. 2). GSK3 is a highly evolutionarily conserved multifaceted ubiquitous enzyme with over 40 different downstream substrates (Wildburger and Laezza, 2012;Jaworski et al., 2019). We next evaluated three proteins known to be regulated by GSK3β -(i) (CaV3.1, (ii) the transcription factor Forkhead box protein P2 (FOXP2), and (iii) the axonal guidance and growth cone collapsin response mediator protein 2 (CRMP2) (Chew and Khanna, 2018;Moutal et al., 2019;Khanna et al., 2012). All three are expressed and have functions in cortical neurons (Morgan-Smith et al., 2014;Bonkowsky et al., 2008;Wildburger and Laezza, 2012;Cheong and Shin, 2013). In agreement with our previous findings (Janakiraman et al., 2019), we found a decrease in CaV3.1 expression in TAF1 edited animals which was reversed by SAK3 treatment (Fig. 7A, B). We observed a decrease of FOXP2 protein levels in TAF1-edited animals which was restored by SAK3 treatment (Fig.  7C, D). Accordingly, while CRMP2 expression was unchanged, its phosphorylation by GSK3β (at T509/T514) was decreased in TAF1 edited animals and rescued by SAK3 treatment (Fig. 7E, F).

Discussion
In the present study, we demonstrated that the T-type channel enhancer SAK3 can efficiently reverse behavioral, morphological, and biochemical defects induced by TAF1 editing in neonatal rats. Pyramidal neurons in layer 5 in the sensory-motor cortex can integrate a large amount of information and propagate signals because of the large dendritic tree and the lengths of their axons; additionally, they are the output neurons of the cerebral cortex and the main source of cognitive and motor development (Jiang et al., 2020). Based on these findings we made the following salient observations: (a) SAK3 treatment improves locomotion defects associated with TAF1 gene editing; (b) SAK3 treatment reduces the number of cortical neurons and astrocytes within the cerebral cortex that were lost following TAF1 editing induced apoptosis; and (c) SAK3 treatment re-activates BDNF/AKT/GSK3β signaling in TAF1 edited animals.
The cerebral cortex and the cerebellum are both involved in movement and there are reciprocal interconnections by the cerebello-thalamo-cortical pathways (Mendoza and Merchant, 2014). The motor cortex is the region within the cerebral cortex important for planning, control, and execution of voluntary movements. In agreement with our previous findings, we observed abnormalities in the somatosensory motor cortex pyramidal neurons in layer 5 (Janakiraman et al., 2019). The somatosensory motor cortex pyramidal neurons in layer 5 are involved in motor signaling and cognitive development. The cerebellum has traditionally been viewed as a brain region involved in motor control, since it receives all the information from the motor cortex, sensory cortex, and spinal cord. However, we present findings that the abnormalities within the cerebral cortex could also contribute to the impairment locomotion seen in gRNA-TAF1 edited animals.
Consistent with our previous findings in the cerebellum, we found that TAF1 editing decreased the number of GFAP positive astrocytic cells (Janakiraman et al., 2019). Astrocytes play essential roles in regulating synaptic connectivity and function during development and in the adult. In development, astrocytes regulate synapse number by secreting synaptogenic signals, such as thrombospondins, and the matricellular proteins hevin, and glypicans, which play a role synapse formation (Allen et al., 2012;Christopherson et al., 2005;Kucukdereli et al., 2011) and by eliminating excess synapses by the phagocytosis receptors MER/AXL/TYRO3 receptor kinase family (MERTK) and Multiple EGF Like Domains 10 (MEGF10) and indirectly by inducing expression of complement cascade components in neurons (Schafer et al., 2013;Chung et al., 2013;Stevens et al., 2007). In the adult brain, astrocytes maintain neuronal and synaptic function, in part via many fine processes that ensheath neuronal synapses (Khakh and Sofroniew, 2015). Functions such as recycling neurotransmitters and buffering extracellular potassium to facilitate neuronal transmission (Allen, 2014) and taking up nutrients, including lipids and sugars from the blood, processing, and delivering them to neurons for energy (van Deijk et al., 2017). SAK3 administration to the TAF1 edited animals increased the number of GFAPpositive astrocytic cells relative to the TAF1 edited group and most likely restores these critical functions within the cerebral cortex.
In agreement with our previous studies, we found morphological abnormalities in cortical neurons in TAF1 edited animals (Janakiraman et al., 2019). In addition, we also observed a marked decrease in cell viability in cerebral cortex at layer 5. The plasticity of the pyramidal neuron morphology in motor cortex are influenced by the cognitive and executive functions (Jiang et al., 2020). To gain insight into the pathogenesis of these abnormalities, we investigated apoptosis. Apoptosis is the most common mode of neuronal death within the central nervous system (Kerr et al., 1972;Kerr et al., 2002). Indeed, we found that apoptosis was increased in TAF1 edited rats by TUNEL and cleaved caspase 3 analysis. Furthermore, we show increased levels of Bax in TAF1 edited animals. There is a plethora of evidence indicating a close relationship between Bax/Bcl-2 during apoptosis (Liu et al., 2011;Kerr et al., 2002;Green and Reed, 1998;Adams and Cory, 2007). Bcl-2 is the primary protein that inhibits cell apoptosis and is expressed in healthy cells, whereas Bax stimulates apoptosis by disrupting mitochondrial function (Adams and Cory, 2007). Hence, our observations suggest that TAF1 gene editing stimulates intrinsic apoptosis pathways in cortical neurons. Moreover, SAK3 treatment mitigates apoptosis caused by TAF1 gene editing.
To further gain insight into signaling pathways involved in the neuroprotective effects of SAK3 treatment against the adverse effects of TAF1 editing, we investigated the BDNF/AKT/GSK3β pathway. Brain-derived neurotrophic factor (BDNF) plays a key role in the pathogenesis of many neurodegenerative disorders (Giacobbo et al., 2019). BDNF binds to tyrosine kinase receptor B to activate Phosphatidylinositol-3-kinase (PI3K) and AKT, thus inhibiting GSK3β activity. We found that the neuroprotective effects of SAK3 treatment are associated with stimulation of in the BDNF/AKT/GSK3 pathway in TAF1 edited animals.
GSK3 has been reported to phosphorylate more than 40 proteins (Wada, 2009) and several studies highlight that GSK3 is an essential mediator of several signaling pathways important in cortical neuron homeostasis (Chen et al., 2008;Renaud et al., 2008;Nakamura et al., 2009). In the cell, two independently regulated GSK3 pools exist-the Wnt signaling pathway and the Wnt independent signaling pathway (Jaworski et al., 2019). Our results suggests that SAK3 treatment activates both pools of GSK3 since we observed an increased levels of CaV3.1 and FOXP2, which are downstream WNT signaling targets (Wisniewska et al., 2010;Bonkowsky et al., 2008). FOXP2 is the fork-head domain transcription factor, mutations of which have been associated with severe deficits in language (Lennon et al., 2007;Feuk et al., 2006;Zeesman et al., 2006). FOXP2 also regulates several downstream target genes that function in synaptic plasticity, neurotransmission, and neurite outgrowth Spiteri et al., 2007). In addition, we observed that SAK3 treatment restored the phosphorylation levels of CRMP2, at Thr509/514, a GSK3β substrate that is regulated independently of Wnt signaling and implicated in cortical neuron radial migration and dendritic orientation (Morgan-Smith et al., 2014). We conclude that regulation of GSK3β is critical for the disease modifying effects of SAK3 treatment.
In conclusion, our findings imply that the T-type calcium channels are novel molecular targets to develop therapeutics to treat TAF1 ID syndrome and that SAK3 is an attractive drug candidate to treat TAF1 associated neurological disorders.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.    The effects of SAK3 treatment on regulators of apoptosis in the cerebral cortex. (A) The effects of SAK3 administration on cleaved caspase 3 levels. Representative Western analysis is shown from cerebral cortex samples from animals from each of the experimental conditions. Note the increased cleaved caspase 3 in gRNA-TAF1 edited animals and diminished cleaved caspase 3 levels in gRNA-TAF1 SAK3 treated animals. (C) Blots show the increased BAX levels in gRNA-TAF1 edited animals, whereas SAK3 reduced BAX levels to control levels. (E) Blots show decreased Bcl-2 levels in gRNA-TAF1 edited animals, whereas SAK3 increase Bcl-2 expression to control levels. (B, D, F) Quantification of Western analysis from independent experiments. Data shown are mean ± SEM, n = 6 animals per each experimental condition. *p < 0.05 versus gRNA-control water and RNAcontrol SAK3 group; # p < 0.05 versus gRNA-TAF1 water group (ANOVA followed by Tukey's test). The effects of SAK3 treatment on BDNF expression in the cerebral cortex of TAF1 edited animals. (A) Expression of BDNF was decreased in TAF1-edited animals (Av) as compared to all other experimental groups (Ai-iv). SAK3 administration to the TAF1 edited animals shows increased number of BDNF-positive cells as compared to TAF1 edited group (Avi & v). (C) Representative Western analysis of BDNF is shown from cerebral cortex samples from animals from each of the experimental conditions. Note decreased BDNF levels in gRNA-TAF1 edited animals, whereas SAK3 increases BDNF expression to control levels. (B) Summary of BDNF expression, Data are shown as mean ± S.E.M., n = 12 fields per animal, 4 animals per experimental condition. Scale bars: 200 μm. (D) Quantification of Western analysis from three independent experiments. Data shown are mean ± SEM, n = 6 animals per each experimental condition. *p < 0.05 versus; naïve and gRNA-TAF1 = SAK3 group (ANOVA followed by Tukey's test). The experiments were conducted in an investigator-blinded manner. The effects of SAK3 treatment on AKT/GSKβ signaling in the cerebral cortex of TAF1 edited animals. (A) Expression of p-AKT was decreased in TAF1-edited animals (Av) as compared to all other experimental groups (Ai-iv). SAK3 administration to the TAF1 edited animals shows increased expression of p-AKT as compared to TAF1 edited group (Avi & v). (C) Representative Western analysis of p-GSK3β is shown from cerebral cortex samples from animals from each of the experimental conditions. Note decreased p-GSK3β levels in gRNA-TAF1 edited animals, whereas SAK3 increases p-GSK3β expression to control levels. (B) Summary of p-AKT expression, Data are shown as mean ± S.E.M., n = 12 fields per animal, 4 animals per experimental condition. Scale bars: 200 μm. (D) Quantification of Western analysis from three independent experiments. Data shown are mean ± SEM, n = 6 animals per each experimental condition. *p < 0.05 versus; naïve and gRNA-TAF1 = SAK3 group (ANOVA followed by Tukey's test). The experiments were conducted in an investigator-blinded manner. However, SAK3 returned the phosphorylation levels of CRMP2, at Thr514 to control levels. (F) Quantification of Western analysis from three independent experiments. Data shown are mean ± SEM, n = 6 animals per each experimental condition. *p < 0.05 versus gRNAcontrol water and RNA-control SAK3 group; # p < 0.05 versus gRNA-TAF1 water group (ANOVA followed by Tukey's test).
Page 22 Table 1 List of primary and secondary antibodies used in the study.

Company Catalog number Application Dilution
Bcl-2-associated X protein