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Novel Function of PIWIL1 in Neuronal Polarization and Migration via Regulation of Microtubule-Associated Proteins

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Novel Function of PIWIL1 in Neuronal Polarization and Migration via Regulation of Microtubule-Associated Proteins

Ping-Ping Zhao et al. Mol Brain.

Abstract

Background: Young neurons in the developing brain establish a polarized morphology for proper migration. The PIWI family of piRNA processing proteins are considered to be restrictively expressed in germline tissues and several types of cancer cells. They play important roles in spermatogenesis, stem cell maintenance, piRNA biogenesis, and transposon silencing. Interestingly a recent study showed that de novo mutations of PIWI family members are strongly associated with autism.

Results: Here, we report that PIWI-like 1 (PIWIL1), a PIWI family member known to be essential for the transition of round spermatid into elongated spermatid, plays a role in the polarization and radial migration of newborn neurons in the developing cerebral cortex. Knocking down PIWIL1 in newborn cortical neurons by in utero electroporation of specific siRNAs resulted in retardation of the transition of neurons from the multipolar stage to the bipolar stage followed by a defect in their radial migration to the proper destination. Domain analysis showed that both the RNA binding PAZ domain and the RNA processing PIWI domain in PIWIL1 were indispensable for its function in neuronal migration. Furthermore, we found that PIWIL1 unexpectedly regulates the expression of microtubule-associated proteins in cortical neurons.

Conclusions: PIWIL1 regulates neuronal polarization and radial migration partly via modulating the expression of microtubule-associated proteins (MAPs). Our finding of PIWIL1's function in neuronal development implies conserved functions of molecules participating in morphogenesis of brain and germline tissue and provides a mechanism as to how mutations of PIWI may be associated with autism.

Figures

Fig. 1
Fig. 1
PIWIL1 regulates cortical radial migration. a-f Effect of knockdown of PIWIL1 in rat cortical neurons by IUE with plasmids coding for PIWIL1 siRNA (RNAi 1). Coronal brain sections at different stages were stained with anti-GFP (green) and DAPI (red). g-l Distribution of labeled cells in brains electroporated with RNAi 1 (g-i) and RNAi 4 (j-l) or control constructs (Scramble). m Different forms of truncated human PIWIL1 (HIWI) or mutated mouse PIWIL1. n-s, u Only constructs containing both PAZ and PIWI domains rescued the migration. The Scramble control data were from the experiment in Additional file 2: Figure S2E. CAG, pCAG-IRES-GFP vector as control. t, u Co-electroporation of D633A-Res with RNAi 2 rescued the migration defect. Scale bar, 250 μm. Error bar, SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test)
Fig. 2
Fig. 2
PIWIL1 is required for the multipolar–bipolar transition of postmitotic neurons. a, d Morphology of labeled neurons in different cortical regions 3 or 5 days post-IUE with siRNA 1. b, e Traces of labeled neurons 3 or 5 days after IUE respectively. c, f Percentage of bipolar cells (white arrows) in different cortical regions. Data are from at least 3 independent IUE experiments. g Typical morphology of labeled mouse neurons in the IZ 3 days after IUE with RNAi 2 or RNAi 2 plus HIWI compared with individual control plasmid. h Percentage of bipolar cells at the IZ of electroporated mouse cortex. Scale bar, 30 μm. Error bar, SEM, **P < 0.01, ***P < 0.001 (Student’s t-test)
Fig. 3
Fig. 3
PIWIL1 knockdown impairs polarization of cortical neurons ex vivo. a Diagram of the ex vivo assay. b, c Average numbers of primary neurites of electroporated cells. d Immunostaining: cultured neurons with PIWIL1 knockdown exhibited multipolar morphology and lower levels of Tau but not Tuj1. e Average neurites’ fluorescence intensity of Tau in GFP+ neurons. Scale bar, 20 μm. Error bar, SEM, *P < 0.05, ***P < 0.001 (Student’s t-test)
Fig. 4
Fig. 4
Identification of PIWIL1 target genes. a Strategy for identifying target genes. b Fold changes of the mRNA levels of several MAPs based on mRNA sequencing results. c, d Western blots showing the reduction of MAP1B, MAP2, and Tau, but not DCX, by PIWIL1 knockdown in cultured cortical neurons. Error bar, SEM. *P < 0.05, **P < 0.01 (Student’s t-test)
Fig. 5
Fig. 5
PIWIL1 affects neuronal migration through regulating the expression of MAPs. a, b MAP2 levels (bands indicated by the arrow) were significantly decreased in the cortical tissue of adult PIWIL1-knockout mice (tissues were all from male animals). Statistics analysis was based on 4 technical repeats on pooled samples (n = 2). c Cortex of PIWIL1-knockout mice showed lower level of MAP2 immunofluorescence signal (red arrows indicate MAP2 signal in WT brain). Scale bar, 20 μm. d, e Co-electroporation of MAP2B with PIWIL1 siRNA largely attenuated the migration defect in mouse brains. Scramble vs RNAi 2; RNAi 2 + CAG vs RNAi 2 + MAP2B. Scale bar, 150 μm. Error bar, SEM, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test)
Fig. 6
Fig. 6
PIWIL1 may regulate MAPs through stabilizing mRNA, but not DNA methylation. a Localization of PIWIL1 in neurons. Dissociated cortical neurons were electroporated with Flag or GFP fused PIWIL1 plasmids (green). PIWIL1 was mainly localized to the cytoplasm, Scale bar, 20 μm. b Western blotting shows that PIWIL1 knockdown decreased the protein level of MAP1B, MAP2, and Tau, but not DCX. Treatment with the DNA methyltransferase inhibitor 5′AZA (2 μM) did not prevent the reduction of MAPs caused by PIWIL1 siRNA. c Neurons were electroporated with Scramble or RNAi plasmid. 48 h later, ActinomycinD (ActD) was added to inhibit transcription for 0, 3 or 6 h. MAP1B mRNA levels were measured by qPCR. The RNA levels at 0 h time point were set as 100 %. Knockdown of PIWIL1 resulted in a faster decay of MAP1B mRNA. Error bar, SEM. d Specific interaction between PIWIL1 and the mRNA of MAP1B. Upper panel, cortical neurons were transfected with Flag-fused HIWI or the vector (CMV-3xFlag). The RNA-protein complex was immunoprecipitated (IP) by anti-Flag antibody. The pull-down of HIWI was validated by Western Blotting, with actin as the indicator of equal input. Weak exposure image indicates that band of IgG group is the non-specific signal with lower molecular weight compared to HIWI-Flag. Lower panel, mRNA of MAP1B immunoprecipitated was revealed by semi-quantitative RT-PCR

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