Disc1 Carrier Mice Exhibit Alterations in Neural pIGF-1Rβ and Related Kinase Expression

Mutation of the disc1 gene underlies a broad range of developmental neuropsychiatric defects, including schizophrenia, depression, and bipolar disorder. The pathophysiological phenotypes linked with disc1 mutation are due to the truncation of the DISC1 primary protein structure. This leads to a defective post-synaptic scaffolding and kinase—GSK3β and Erk1/2—signaling. As a result, synaptic function and maintenance are significantly impaired in the disc1 mutant brain. Among several other pathways, GSK3β and Erk1/2 are involved in insulin-like growth factor 1 receptor (IGF-1Rβ) kinase signaling. Although disc1 mutation alters these kinases, it is unclear if the mutation impacts IGF-1R expression and activity in the brain. Here, we demonstrate that the expression of active IGF-1Rβ (pIGF-1Rβ) is altered in the hippocampus and prefrontal cortex (PFC) of disc1 mutant mice and vary with the dose of the mutation (homozygous and heterozygous). The expression of pIGF-1Rβ decreased significantly in 129S (hom, disc1−/−) brains. In contrast, 129S:B6 (het, disc1+/−) brains were characterized by an increase in pIGF-1Rβ when compared with the C57BL/6 (disc1+/+) level. The decrease in pIGF-1Rβ level for the 129S brains was accompanied by the loss of Akt activity (S473 pAkt) and decreased Ser9 phosphorylation of GSK3β (increased basal GSK3β). Additionally, hippocampal and cortical pErk1/2 activity increased in the 129S hippocampus and cortex. Although 129S:B6 recorded alterations in pIGF-1Rβ-pAkt-GSK3β (like 129S), there was no observable change in pErk1/2 activity for the heterozygote (disc1+/−) mutant. In addition to GSK3β inhibition, we conclude that pIGF-1R, pAkt, and pErk1/2 are potential targets in disc1−/− mutant brain. On the other hand, pIGF-1R and pAkt can be further explored in disc1+/− brain.

Although disc1 mutation promulgates erroneous GSK3β and Erk1/2 activity, the impact on pIGF-1Rβ expression and activity is yet to be investigated in the cognitive centers. Erk1/2 and GSK3β are downstream effector molecules of pIGF-1Rβ kinase activity and are involved in the maintenance of the synaptic structure. GSK3β and Erk1/2 activity are also pertinent to the propagation of LTP, and coupling of synaptic function to cellular regulation (Peineau et al., 2007;Dewachter et al., 2009;Vara et al., 2009;Giachello et al., 2010;Shahab et al., 2014). Downstream of pIGF-1Rβ, Erk1/2 (Roux and Blenis, 2004;Roskoski, 2012) and GSK3β (Hur and Zhou, 2010) are involved related pathways that regulates cell proliferation and cell survival. As such, alteration in the activity of these kinases in disc1 mutation may disrupt signaling cascades that involve pIGF-1Rβ.
The study provides evidence of pIGF-1Rβ dysregulation in the hippocampus and prefrontal cortex (PFC) of mutant disc1 carrier mice. In addition to changes in neural GSK3β and Erk1/2 expression, heterozygous 129S:B6 (disc1 +/− ) and homozygous 129S (disc1 −/− ) carriers exhibit a change in neural pIGF-1Rβ expression. Here, we show some of the differences and similarities in the pattern of pIGF-1Rβ dysregulation for the hippocampus and PFC of these disc1 carrier mice.

MATERIALS AND METHODS
The 129S (disc1 −/− ) mice (RRID:IMSR_JAX:002448) were acquired from the Jackson Lab (Bar Harbor, ME, United States) and have a spontaneous C-terminal truncation mutation in the disc1 gene (Clapcote and Roder, 2006). The 129S:B6 (disc1 +/− ) line (RRID:IMSR_JAX:101043) is from a cross of the 129S and C57BL/6J mouse lines. For comparison, we used the C57BL/6J (B6) line (RRID:IMSR_JAX:000664) as carriers of the wildtype disc1 gene (disc1 +/+ ). We have previously demonstrated that 129S mice vary behaviorally from all other inbred strains, including the C57BL/6J, and have phenotypes that are similar to other disc1 knockout strains (Sultana et al., 2019). Animals were housed under standard laboratory conditions of 12 h alternating light and dark cycle with food and water provided ad libitum. All animal handling procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Louisiana State University School of Veterinary Medicine. Adult mice (PND 90-100) weighing between 22-26 g were used for this study (C57BL/6J: n = 9, 129S:B6: n = 9; 129S: n = 10).

Specimen Preparation
Mice were euthanized in an isoflurane chamber. Subsequently, the animals were transcardially perfused with 10 mM PBS (pH 7.4). The whole brain was harvested and rapidly placed in cold artificial cerebrospinal fluid (aCSF) maintained on ice, and saturated with 95% Oxygen/5%CO 2 . A clean razor blade was used to cut the brain-along the sagittal plane-into two (left and right) hemispheres. The left and right hemispheres were microdissected, and the hippocampus was extracted by exposing the space between the cortex and corpus callosum. A surgical blade was used to cut the PFC. The harvested hippocampal and prefrontal cortical tissue was kept in separate tubes and stored at −80 • C until further use.

Quantification
Fluorescence imaging was performed using a Nikon-NiU fluorescence upright microscope configured for 3D imaging. Z-stacks were obtained and converted into 2D images through the extended depth focus (EDF) option on Nikon Element software. Normalized fluorescence intensity for immunolabeled proteins in the hippocampus and medial PFC was performed in optical slices for serial section images (n = 5 per group). Fluorescence intensity was quantified using Nikon Element AR. Mean cell count and intensity were determined per unit area in several fields of view for consecutive sections. Fluorescence intensity was normalized by applying a uniform exposure time for a fluorophore-labeled protein in the control and test brain slices.
Subsequent analysis of Akt, GSK3β, and Erk1/2 expression showed that a decreased pIGF-1Rβ level in the 129S brain may be related to DISC1 loss of function. As such, in the disc1 −/− brain, a decrease in pIGF-1Rβ was accompanied by a loss of pAkt (Ser473), increased basal GSK3β activity, and a general increase in pErk1/pErk2 activity.

pAkt
The kinase activity of pIGF-1Rβ involves the downstream activation of Akt (PBK) through Thr308 phosphorylation (Figure 1). Given that the complete activation of Akt requires Ser473 phosphorylation, here, we evaluated the expression of pAkt (S473) in C57BL/6J, 129S:B6 and 129S brains. Akt expression was determined by normalizing the basal protein level with β-actin. The threshold of Akt activity was determined by normalizing S473 phosphorylated Akt with Akt protein level. Partial (disc1 +/− ) and total (disc1 −/− ) ablation of DISC1 function did not alter the basal Akt level in the 129S hippocampus ( Figure 4A). As a result, no significant change was recorded for Akt expression when 129S was compared with 129S:B6 and B6. Similarly, Akt expression in the 129S:B6 hippocampus did not change significantly vs. the B6 levels ( Figure 4B). Although the disc1 ablation did not impact Akt expression, subsequent analysis of Akt activity level revealed otherwise ( Figure 4C). The loss of DISC1 function in the 129S hippocampus significantly reduced S473 phosphorylation of Akt when compared with the control (B6; p < 0.01). Although the 129S:B6 did not record a decline in pIGF-1R activity or Akt expression, the disc1 +/− phenotype was also characterized by a reduction of S473 pAkt; compared with B6 level (p < 0.05). As such, there was no significant difference in normalized hippocampal S473 pAkt when 129S was compared with 129S:B6 level. Furthermore, both mutant phenotypes (disc1 +/− and disc1 −/− ) recorded a significant loss of S473 pAkt when compared with the B6 (disc1 +/+ ). Based on these outcomes, loss of S473 pAkt activity in the 129S (disc1 −/− ) hippocampus may be directly linked to the decline in hippocampal pIGF-1Rβ activity. However, since the pIGF-1Rβ activity did not reduce in the 129S:B6 hippocampus, loss of S473 pAkt activity might have occurred as a result of other changes directly linked to a defective DISC1 signaling.

GSK3β
GSK3β is involved in several cellular processes that occur downstream of IGF-1Rβ and other tyrosine kinase receptors (Rtks). Unlike IGF-1Rβ and Akt, GSK3β is basally active and does not require phosphorylation to be activated (Dewachter et al., 2009;Hur and Zhou, 2010;Emamian, 2012;Bhat et al., 2018). Mechanistically, phosphorylation of GSK3β (pGSK3β) at the Ser9 site by S473 pAkt attenuates basal GSK3β activity. Here, we used western blotting to detect basal GSK3β expression, and the threshold of Ser9 GSK3β phosphorylation in the hippocampus and PFC. To determine basal GSK3β level, GSK3β was normalized with β-actin. Likewise, the threshold of Ser9 GSK3β phosphorylation was determined by normalizing Ser9 pGSK3β with basal GSK3β.
Immunoblot analysis of the prefrontal cortical whole lysate revealed a significant increase in basal GSK3β activity for the 129S:B6 (p < 0.05) and 129S (p < 0.05) brain (Figures 5H,I).
Contrary to the hippocampus, the 129S:B6 cortex showed an increase in normalized Ser9 pGSK3β level (p < 0.01; Figure 5I) vs. the C57BL/6J. This indicates a decreased GSK3β activity in the 129S:B6 PFC compared with the control (B6). Interestingly, for the 129S PFC, there was no significant change in Ser9 pGSK3β level vs. the C57BL/6J (Figures 5G-I). From these outcomes, we determine that an increase in prefrontal cortical basal GSK3β activity is a shared attribute of 129S:B6 and 129S PFC. However, the pattern of cortical dysregulation of Ser9 pGSK3β may be dose-specific.
Immunoblot analysis of whole PFC lysate showed a significant increase in basal Erk1/2 protein level for the 129S:B6 (p < 0.05) and 129S (p < 0.05) PFC (Figures 6H,I). Analysis of pErk1 and pErk2 activity in the cortex showed some variations when compared with the hippocampus. Similar to the hippocampus, there was no significant change in total pErk1 activity for the 129S PFC; compared with the B6 and 129S:B6 ( Figure 6J). Interestingly, the 129S PFC recorded a significant increase in pErk2 activity when compared with B6 and 129S:B6 (p < 0.01; Figure 6K). These outcomes suggest that disc1 −/− mutation impacts pErK2 function in the PFC and not pErk1.
Our results also showed that the heterozygote disc1 mutation (129S:B6) did not impact pErk1/2 activity in the hippocampus and PFC. As such, there was no significant difference in normalized pErk1 and pErk2 levels when we compared 129S:B6 to B6 hippocampus (Figures 6A,B). This was also the case for the synaptic activity of pErk1 and pErk2 (Figures 6D-G). Similarly, prefrontal cortical expression of pErk1 and pErk2 were not significantly different when the 129S:B6 (disc1 +/− ) was compared to B6 (disc1 +/+ ; Figures 6H,K,L).
Erroneous regulation of Erk1/2 signaling promulgates cell proliferation abnormalities in the developing cortical circuit. Experimental hyperactivation of Erk1/2 in the cortex caused a significant increase in neuron count, and morphological defects (Morales-Garcia et al., 2014;Xing et al., 2016). To this effect, pharmacological inhibition of Erk1/2 signaling rescued some of the synaptic and behavioral phenotypes associated with developmental neuropsychiatric defects in mice (Lu and Dwyer, 2005;Soares et al., 2011;Pereira et al., 2014;Tassin et al., 2015;Aringhieri et al., 2017;Hirayama-Kurogi et al., 2017;Pucilowska et al., 2018). Similarly, there is evidence that pharmacological inhibition or genetic ablation of neural GSK3β activity rescued dendritic spine and behavioral abnormalities linked to disc1 mutation, and other forms of schizophrenia (Lee et al., 2011b;Lipina et al., 2011).
In addition to their role in DISC1 signaling, GSK3β and Erk1/2 are controlled by upstream pIGF-1Rβ activity. An important question yet to be addressed is whether a change in expression of these kinases in the disc1 mutant brain impacts pIGF-1Rβ activity. Based on previously established mechanisms for pIGF-1Rβ signaling (Dyer et al., 2016), we considered the possible link between IGF-1Rβ, Akt, Erk1/2, GSK3β in the disc1 mutant brain. In addition to a change in the activity of GSK3β and Erk1/2, neural pIGF-1R expression changed in the disc1 mutant hippocampus and PFC. Analysis of pIGF-1Rβ-GSK3β signaling showed that the decrease in neural pIGF-1Rβ in the 129S hippocampus was accompanied by the suppression of S473 pAkt. Likewise, the loss of S473 pAkt may be linked to a decrease in Ser9 phosphorylation (inactivation) of pGSK3β (Wan et al., 2007;Kitagishi et al., 2012;Levenga et al., 2017). From these outcomes, we deduced that the dysregulation of DISC1-associated proteins (GSK3β/Erk1/2) impacts pIGF-1Rβ activity in the disc1 mutant brain (Figure 1).
Our results revealed that an increase in basal GSK3β activity, in the hippocampus of mutant disc1 carrier mice, is associated with the dysregulation of DISC1-GSK3β interaction, and pAkt-mediated regulation of GSK3β. Thus, in both disc1 +/− and disc1 −/− brain, there was a decrease in Ser473 pAkt and a reduction in Ser 9 phosphorylation of GSK3β. This indicates an increase in basal GSK3β activity. A similar increase in GSK3β activity was also observed at the synaptic level. Accordingly, 129S:B6 and 129S synaptosomal extracts recorded a decrease in normalized Ser9 pGSK3β level when compared with the control (B6; Figures 5E,F). Like the hippocampus, the PFC of mutant disc1 carriers also exhibits a significant increase in basal GSK3β expression when compared with the control (B6; Figure 5I). However, the pattern of GSK3β activity was different when the hippocampus was compared with the cortex. While the 129S:B6 (disc1 +/− ) showed an increase in normalized Ser9 pGSK3β, the 129S (disc1 −/− ) PFC recorded no significant change vs. the control.
The observed prefrontal cortical GSK3β activity may be related to the dysregulation of pIGF-1Rβ-pAkt in the mutant cortex. As shown previously, the disc1 +/− (129S:B6) brain was characterized by an increase in pIGF-1Rβ expression (Figures 3D-F). When compared with the control (B6), the level of normalized S473 pAkt was also unchanged for the 129S:B6 cortex ( Figure 4F). Thus, there is a possibility that an increase in pIGF-1Rβ expression promulgates a higher level of Ser9 pGSK3β in the 129S:B6 cortex, compared with the 129S. Interestingly, the loss of pIGF-1Rβ in the disc1 −/− (129S) cortex ( Figure 3D-F), coupled with a decrease in S473 pAkt ( Figure 4F) did not affect the Ser9 pGSK3β threshold. These results suggest that change in Ser9 pGSK3β in the 129S (disc1 −/− ) PFC may not be directly linked to pIGF-1Rβ dysregulation.
In addition to a defective pIGF-1Rβ-pAkt-GSK3β, Erk1/2 activity is also dysregulated in the hippocampus and PFC of 129S (disc1 −/− ) mutant mice. Specifically, there was an increase in pErk1/2 activity in the hippocampus and pErk2 activity in the PFC of 129S mice. Interestingly, increased ErK activity appeared to phenotype-specific. While the 129S (disc1 −/− ) recorded an increased synaptic pErk level, there was no significant change in pErk activity for the 129S:B6 (disc1 +/− ) brain when compared with the control (B6; Figure 6). Given that a pIGF-1Rβ decreased in 129S hippocampal whole lysate and synaptosomal extracts, it is likely that pErk increase may be associated with other changes linked to disc1 mutation and post-synaptic DISC1 activity. Similarly, increased pErk2 levels in the 129S PFC demonstrate that a change in Erk activity may not be dependent on pIGF-1Rβ. The limitation of the current study is that loss or gain of function experiments have not been performed for Erk, GSK3β, and Akt. Thus, the results are still preliminary and descriptive.
Taken together, these outcomes suggest that the hippocampus and PFC show distinct patterns of kinase dysregulation in disc1 mutation. Also, the pattern of dysregulation may vary with the mutation dose. While the 129S:B6 showed mostly GSK3β dysregulation, the 129S brain recorded significant changes in GSK3β and Erk1/2.

FUTURE DIRECTIONS
Although Erk1/2 and GSK3β inhibitors are now being explored as therapeutic agents in developmental neuropsychiatric disorders, our results suggest that a broader target involving pIGF-1Rβ may be required for effective control of the pathway. The outcome of this study also revealed a general decrease in neural pIGF-1Rβ as a result of disc1 mutations (Figures 1, 2). This is in agreement with previous studies that explored IGF-1 therapy to attenuate schizophrenia pathophysiology (Venkatasubramanian et al., 2010;Bou Khalil, 2011;Demirel et al., 2014). On the other hand, an increase in pIGF-1Rβ in the 129S:B6 brain suggests that IGF-1Rβ blockers could be explored as therapeutic targets for some disc1 carriers. In addition to an increase in GSK3β activity, pErk1/2 is also upregulated in the 129S brain. Thus, a combination of GSK3β and Erk1/2 inhibitors could be further explored.

SUMMARY
In summary, our results show that neural pIGF-1Rβ expression is altered in 129S:B6 and 129S mice compared with C57BL/6J animals. Furthermore, both strains were characterized by a significant change in GSK3β and Erk1/2 expression patterns in the hippocampus and PFC (Supplementary Table S1). We deduce that some of these changes may be directly related to pIGF-1Rβ expression. Thus, targeting IGF-1Rβ in addition to the kinases (GSK3β and Erk1/2) may reduce the phenotypic burden of some developmental neuropsychiatric disorders.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
The animal study was reviewed and approved by Louisiana State University School of Veterinary Medicine IACUC Committee.