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Research Article
Koh-ichi Nagata
Nagoya University
Corresponding Author
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WDR45 plays an essential role in the early stage of autophagy. De novo heterozygous mutations in WDR45 have been known to cause b-propeller protein-associated neurodegeneration (BPAN), a subtype of neurodegeneration with brain iron accumulation (NBIA). Although BPAN patients display global developmental delay including intellectual disability, neurodevelopmental pathophysiology of BPAN remains largely unknown. In the present study, we analyzed the physiological role of Wdr45 and pathophysiological significance of the gene abnormality during mouse brain development. Morphological and biochemical analyses revealed that Wdr45 is expressed in a developmental stage-dependent manner in mouse brain. Wdr45 was also found to be located in the excitatory synapses in biochemical fractionation. Since the WDR45 mutations are thought to cause protein degradation, we conducted acute knockdown experiments by an in utero electroporation method with mice to recapitulate the pathophysiological conditions of BPAN. Silencing of Wdr45 caused abnormal dendritic development and synaptogenesis during corticogenesis, both of which were significantly rescued by co-expression with RNAi-resistant version of Wdr45. In addition, terminal arbors of callosal axons were less developed in Wdr45-deficient cortical neurons of adult mouse when compared to the control cells. These results strongly suggest a pathophysiological significance of WDR45 gene abnormalities in neurodevelopmental aspects of BPAN.
Keywords
WDR45, corticogenesis, dendrite arborization, axon pathfinding, spine formation
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- SupplementaryDataset2.pdf
Supplementary Figure 1. Immunohistochemical analyses of Wdr45 in hippocampus and cerebellum Hippocampal sections at E17 (A), P0 (B), P7 (C) and P30 (D) were stained with anti-WDR45. Boxed areas in (a) were magnified in (b) – (d). Boxed areas in (b) – (d) were magnified in (b’) – (d’), respectively. CA, cornu ammonis; DG, dentate gyrus. (E) A cerebellar section at P30 was stained with anti-WDR45. Boxed areas in (a) and (b) were magnified in (b) and (b’), respectively. Layers were indicated in (b). ML, molecular layer; PCL, Purkinje cell layer; GL, granular layer. Counter staining was done with Mayer’s hematoxylin. Scale bars; 100 µm (A-E,a), 50 µm (A-D,b-d and E,b) and 10 µm (A-D,b’-d’ and E,b’). Supplementary Figure 2. Effects of Wdr45-knockdown on cortical neuron migration during corticogenesis. pCAG-TurboRFP was co-electroporated with pSuper-H1.shLuc (control) or pSuper-Wdr45#1 or #2 into the VZ progenitors at E14.5. Coronal sections were prepared at P2 and stained for RFP. Dotted lines represent the pial (upper) and the ventricular (lower) surfaces. Supplementary Figure 3. Effects of Wdr45-knockdown on LC3 expression. pCAG-TurboRFP was co-electroporated with pSuper-H1.shLuc (Control) or pSuper-Wdr45#1(KD#1) into the VZ progenitors at E14.5. Coronal sections were prepared at P2 and stained for Wdr45 and LC3. Representative images of dendritic spines on the 1st-order apical dendrites were shown. Scale bars, 5 m.
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This preprint is under consideration at Scientific Reports. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Koh-ichi Nagata
Nagoya University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Revision
Version 1
Posted 30 Dec, 2020
No community comments so far
Editorial decision: Major revision
On 06 Apr, 2021
Reviews received
Received 16 Mar, 2021
Reviewers agreed
On 05 Mar, 2021
Reviewers agreed
On 20 Feb, 2021
Reviewers invited
Invitations sent on 14 Feb, 2021
Editor assigned
On 08 Feb, 2021
Editor invited
On 28 Dec, 2020
Submission checks complete
On 28 Dec, 2020
First submitted
On 21 Dec, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
WDR45 plays an essential role in the early stage of autophagy. De novo heterozygous mutations in WDR45 have been known to cause b-propeller protein-associated neurodegeneration (BPAN), a subtype of neurodegeneration with brain iron accumulation (NBIA). Although BPAN patients display global developmental delay including intellectual disability, neurodevelopmental pathophysiology of BPAN remains largely unknown. In the present study, we analyzed the physiological role of Wdr45 and pathophysiological significance of the gene abnormality during mouse brain development. Morphological and biochemical analyses revealed that Wdr45 is expressed in a developmental stage-dependent manner in mouse brain. Wdr45 was also found to be located in the excitatory synapses in biochemical fractionation. Since the WDR45 mutations are thought to cause protein degradation, we conducted acute knockdown experiments by an in utero electroporation method with mice to recapitulate the pathophysiological conditions of BPAN. Silencing of Wdr45 caused abnormal dendritic development and synaptogenesis during corticogenesis, both of which were significantly rescued by co-expression with RNAi-resistant version of Wdr45. In addition, terminal arbors of callosal axons were less developed in Wdr45-deficient cortical neurons of adult mouse when compared to the control cells. These results strongly suggest a pathophysiological significance of WDR45 gene abnormalities in neurodevelopmental aspects of BPAN.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
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