ErbB dysregulation impairs cognition via myelination-dependent and - independent oligodendropathy

White matter abnormalities are an emerging pathological feature of schizophrenia. However, their attributions to the disease remain largely elusive. ErbB receptors and their ligands, some of which are essential for peripheral myelination, confer susceptibility to schizophrenia. By synergistically manipulating ErbB receptor activities in a oligodendrocyte-stage-specific manner in mice after early development, we demonstrate the distinct effects of ErbB signaling on oligodendrocytes at various differentiation states. ErbB overactivation, in mature oligodendrocytes, induces necroptosis causing demyelination, whereas in oligodendrocyte precursor cells, induces apoptosis causing hypomyelination. In contrast, ErbB inhibition increases oligodendrocyte precursor cell proliferation but induces hypomyelination by suppressing the myelinating capabilities of newly-formed oligodendrocytes. Remarkably, ErbB inhibition in mature oligodendrocytes diminishes axonal conduction under energy stress and impairs working memory capacity independently of myelin pathology. This study reveals the etiological implications of oligodendrocyte vulnerability induced by ErbB dysregulation, and elucidates the pathogenetic mechanisms for variable structural and functional white matter abnormalities.


Introduction 1
Adolescence is the critical period for the central nervous system (CNS) to completely 2 develop and mature. In particular, CNS myelin generated by oligodendrocytes (OLs) 3 is one of the most developmentally active component in the adolescent brain. This 4 may lead to CNS myelin being a highly susceptible target in psychiatric disorders 5 such as schizophrenia which typically develops during adolescence (Fields, 2008;6 Hoistad et al., 2009;Kessler et al., 2007;Peters and Karlsgodt, 2015). A growing  Winterer et al., 2008;Zuliani et al., 2011). 34 Notably, in addition to NRG1 and ErbB4, many molecules in the ErbB signaling . It is noteworthy that EGFR (ErbB1), which only binds the EGF family 8 dysfunction, including ataxia and tremors, and died around P35. As a result,  ErbB2 V664E and littermate control mice were investigated at P30 after 9 days with 123 Dox-feeding (dwd). These mice had smaller body sizes at P30 and walked with 124 difficulty on a grid panel ( Figure 1M). Western blotting revealed the expression and 125 phosphorylation of ectopic ErbB2 V664E accompanied with increases in 126 phosphorylation of ErbB3 and ErbB4, but not that of EGFR, in the white matter of 127 Sox10-ErbB2 V664E mice ( Figure 1N   Plp-tTA targets mainly MOs whereas Sox10 +/rtTA targets OPC-NFOs 148 The finding that Plp-ErbB2 V664E and Sox10-ErbB2 V664E mice had no overlaps in 149 histological and biochemical phenotypes was unexpected considering that Sox10 is 150 reported to express throughout the OL lineage, and Sox10 +/rtTA knock-in mice have 151 been used to investigate all OL lineage cells (Wegener et al., 2015). Because the 152 induction of the 'Tet-on' or 'Tet-off' system by Dox feeding or Dox withdrawal has a 153 delayed effect on gene expression, and reporter proteins could accumulate to label the 154 consecutive cellular stages, the results obtained by using TRE-controlled reporter 155 mice fail to accurately reveal the original cells targeted by tTA or rtTA. To 156 circumvent this problem, we delivered a TRE-controlled fluorescence reporter carried 157 by an adeno-associated virus (AAV) into the mouse brains at P14 or P35, and 158 examined tTA-or rtTA-targeted cells as well as their derivatives within 2 days.

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There are also OL lineage cells belonging to the NFO stage that includes a 176 transition from CC1to CC1 + . β-catenin effector TCF4 is specifically expressed in the 177 NFO stage (Fancy et al., 2009;Fu et al., 2009;Ye et al., 2009), which is present in a 178 subset of Olig2 + cells but is absent in OPCs (PDGFRα + ) in mice at P30 ( Figure 2D).

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Interestingly, in Sox10 +/rtTA mice at P35, YFP + cells were mostly found in regions 184 with TCF4 + cell clusters (Figure 2-figure supplement 1E), where approximately 56% 185 of YFP + cells were TCF4 + 1 day after virus injection and it reduced to 29% after 1 186 more day ( Figure 2F,G). The half reduction of NFO percentage in YFP + cells from 187 day 1 to day 2 was consistent with the previous report that NFOs differentiate into 188 MOs in 1 or 2 days (Xiao et al., 2016). There was another possibility that the 189 transcriptional activity of Sox10-rtTA was low in MOs, and thus took more days to 190 generate detectable YFP levels. We analyzed the densities of TCF4 + YFP + cells and 191 found that they reduced to half from day 1 to day 2 after viral infection in Sox10 +/rtTA 192 mice at either P14 or P35 ( Figure 2H). The results excluded the possibility that the 193 reduction of NFO ratio in YFP + cells was due to the increase of targeted MOs, and 194 confirmed the maturation of labeled NFOs from day 1 to day 2. A similar transition 195 11 rate is applicable for targeted NFOs from day 0 to day 1. Therefore, these results 196 corroborate that the majority of cells targeted by Sox10-rtTA at the time of AAV-197 TRE-YFP infection were tOPCs and NFOs ( Figure 2I).

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AAV-TRE-YFP in Plp-tTA mice also labeled some TCF4 + cells, which 199 comprised only 7-12% of YFP + cells 1 or 2 days after virus injection at either age 200 ( Figure 2E-H). It was noticeable that Plp-tTA did not specifically target TCF4 + cell 201 clusters in the corpus callosum at P35 (Figure 2-figure supplement 1F). These results 202 implied that Plp-tTA did not specifically target the tOPC or NFO stage but randomly 203 expressed in the OPC-NFO period at a low ratio. Conversely, 90% of the YFP + cells 204 were TCF4and 92-97% were CC1 + in Plp-tTA mice at either P14 or P35, suggesting 205 that Plp-tTA steadily targets MOs after early development ( Figure 2I).   assay, and observed as little apoptotic signaling as that in controls ( Figure 3D,E). This 223 result reveals that the degenerating CC1 + cells were necrotic rather than apoptotic.       different results from the two mouse lines implied that the impaired motor 365 coordination could be attributed to the hypomyelination in the CNS. 366 We further tested these mice in the eight-arm radial water maze, a paradigm 367 analyzing working memory capacity. It is known that myelin integrity is fundamental  stimulating inflammatory pathological responses in the brain (Figures 1 and 3).                1K. After a trial that mice reached a hidden platform, mice were returned to their 855 home cages with towel and warming pads. There was a 30-sec gap between trials, and 856 the visited platform was removed before the next trial. Mice aborted swimming 857 during training were discarded. Two weeks later, the trained mice were tested for their 858 working memory capacities that were represented by avoiding arms with visited 859 platforms in previous trials. In the last trial of the test day, the animals had highest 860 working memory load for they had to avoid swimming into three arms with platforms 861 removed. First and repeat entries into any arm that previously had a platform were 862 counted as working memory errors, and first entries into any arm that never had a 863 platform were counted as reference memory errors which represent deficits in spatial 864 recognition or long term memory. The day after test day, all the animals were tested 865 in a simple visible platform task with 5 trials in a round pool, and each trial contained 866 a visible platform placed at a different position 0.5-1.0 meter away from tested mice.

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The latency of mice reached the visible platform in each trial was averaged to assess 868 their eyesight.     unpaired Student's t test was used for analysis between two groups with one variable, 941 one-way ANOVA test was used for analysis among three or more groups with one 942 variable, and two-way ANOVA test was used to determine difference among groups 943 42 with two variables. Statistical significance was set at *P < 0.05, **P < 0.01, ***P < in the corpus callosum of indicated mice. Data were presented as mean ± s.e.m. and 1213 analyzed by two-tailed unpaired t test. In F, for the middle part at 9 dpd, t (6) = 6.345, P 1214 = 0.00072; for the lateral part at 9 dpd, t (6) = 3.914, P = 0.0079; for the middle part at