The E3 Ubiquitin Ligase CRL5 Regulates Dentate Gyrus Morphogenesis, Adult Neurogenesis, and Animal Behavior - PubMed (original) (raw)
The E3 Ubiquitin Ligase CRL5 Regulates Dentate Gyrus Morphogenesis, Adult Neurogenesis, and Animal Behavior
Raenier V Reyes et al. Front Neurosci. 2022.
Abstract
The dentate gyrus (DG) is an essential part of the hippocampal formation and participates in the majority of hippocampal functions. The DG is also one of the few structures in the mammalian central nervous system that produces adult-born neurons and, in humans, alterations in adult neurogenesis are associated with stress and depression. Given the importance of DG in hippocampal function, it is imperative to understand the molecular mechanisms driving DG development and homeostasis. The E3 ubiquitin ligase Cullin-5/RBX2 (CRL5) is a multiprotein complex involved in neuron migration and localization in the nervous system, but its role during development and in the adult DG remain elusive. Here, we show that CRL5 participates in mossy fiber pruning, DG layering, adult neurogenesis, and overall physical activity in mice. During DG development, RBX2 depletion causes an overextension of the DG mossy fiber infrapyramidal bundle (IPB). We further demonstrate that the increased activity in Reelin/DAB1 or ARF6 signaling, observed in RBX2 knockout mice, is not responsible for the lack of IPB pruning. Knocking out RBX2 also affects granule cell and neural progenitor localization and these defects were rescued by downregulating the Reelin/DAB1 signaling. Finally, we show that absence of RBX2 increases the number neural progenitors and adult neurogenesis. Importantly, RBX2 knockout mice exhibit higher levels of physical activity, uncovering a potential mechanism responsible for the increased adult neurogenesis in the RBX2 mutant DG. Overall, we present evidence of CRL5 regulating mossy fiber pruning and layering during development and opposing adult neurogenesis in the adult DG.
Keywords: CRL5; RBX2; adult neurogenesis; dentate gyrus development; mossy fibers.
Copyright © 2022 Reyes, Hino, Canales, Dickson, La Torre and Simó.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Figures
FIGURE 1
RBX2 participates in mossy fiber IPB pruning. (A) IPB overextension in the RBX2cKO-Emx1 mice at P21. Stainings of control and RBX2cKO-Emx1 DG showed a similar extension of mossy fiber SPB (arrows demarcate SPB extension), whereas mossy fiber IPD was ectopically extended in the RBX2 mutant DG (arrowheads demarcate IPB extension). (B) Quantification of IPB extension in control, RBX2cKO-Emx1, and SOCS7 knock out (–/–) DG at P21 (left) and P75 (right). Mean ± SEM. Statistics, one-way ANOVA with Tukey’s method adjusted _p_-value. (C) Axons from E18.5 control and RBX2 mutant DG explant were repelled when confronted with a source of Semphorin-3F (Sema3F). Notice the HEK293T cell aggregate transfected with control plasmid or Sema3F-expressing plasmid on the left hand side of the image. For quantification, DG explants were divided in four quadrants and β-III-Tubulin fluorescent signal measured in proximal (in front of the cell aggregate) and distal (opposite) quadrants. (D) The ratio of proximal and distal (p/d) showed that both control and Rbx2 mutant DG axons were repelled by Sema3F. Mean ± SEM. Statistics, unpaired Student’s _t_-test. (E) Decreased RAC1-GTP and CDC42-GTP levels upon RBX2 depletion in comparison to control. RHO-GTP levels remained unaffected.
FIGURE 2
RBX2 regulates layering during DG development and controls NSC proliferation. (A,B) Depletion of RBX2 disrupted the localization of Doublecortin (DCX)+ (arrowheads) and calbindin (CalB)+ cells in comparison to control DG at P21. Similarly, SOX2+ NSCs, which normally locate in the subgranular zone (SGZ), were displaced to other areas of the DG upon RBX2 depletion. (C) Reducing DAB1 accumulation partially rescued the localization defects of DCX+ cells caused by knocking out RBX2, whereas it had little effect in CalB+ and SOX2+ cells. (D) Knocking out SOCS7 did not affect the neuron localization and layering in the DG. Total number (top) and layer distribution (bottom) of SOX2+ (E), DCX+ (F), and CalB+ (G) cells in the DG. The DG was divided in four layers as shown in (A). Mean ± SEM. Statistics, one-way ANOVA with Tukey’s method adjusted _p_-value for total number of cell analyses and two-way ANOVA with Tukey’s method adjusted _p_-value for layering analyses. ML, molecular layer; GCL, granule cell layer.
FIGURE 3
RBX2 controls NSC proliferation in a Reelin/Dab1-dependent fashion. (A,B) Knocking out RBX2 promoted NSC proliferation, detected by Ki-67 staining (arrows), in comparison to control DG at P21. Arrowheads indicate Ki-67+ cells away from the innermost layer in the DG (C) reducing DAB1 levels in the RBX2 mutant DG rescues cell proliferation. (D) SOCS7 depletion showed the same levels of Ki-67+ cells (arrows). (D–F) Quantification of Ki-67+ cells in control (Rbx2 fl/fl) vs. RBX2cKO-Emx1 DG at P10 (E), P21 also including RBX2cKO-Emx1; DAB1 +/– quantification (F), P75 (G), and of control (SOCS7+/–) and SOCS7 mutant (SOCS7–/–) DG at P21 (H). Mean ± SEM. Statistics, unpaired Student’s _t_-test (E,G,H) and one-way ANOVA with Tukey’s method adjusted _p_-value (F).
FIGURE 4
In an open field test, both RBX2 mutant male (A) and female (B) mice (P75) showed increased horizontal (distance moved) and vertical (rearing) activity as well as higher total activity and distance moved in 30 min in comparison to control mice. On the contrary, both male and female RBX2cKO-Emx1 mice did not show changes in the amount of time spent at the center of the field. Mean ± SEM. Statistics, two-way ANOVA for all quantifications, except unpaired Student’s _t_-test for total distance quantification.
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