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Novel function of PIWIL1 in neuronal polarization and migration via regulation of microtubule-associated proteins.

Zhao PP, Yao MJ, Chang SY, Gou LT, Liu MF, Qiu ZL, Yuan XB - Mol Brain (2015)

Bottom Line: Furthermore, we found that PIWIL1 unexpectedly regulates the expression of microtubule-associated proteins in cortical neurons.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.

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.

No MeSH data available.


Related in: MedlinePlus

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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Fig6: 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

Mentions: Considering the well-established roles of microtubule dynamics in neuronal polarization, we next focused on the microtubule-associated proteins which may be a potential target of PIWIL1. As shown in Fig. 4b, we found that in the RNAi-treated group the mRNA levels of several microtubule-associated proteins (MAPs), including MAP1B, MAP2, and Tau decreased significantly. Down-regulation of Tau is consistent with a low immunostaining signal of Tau in cultured newborn neurons that had been electroporated with the PIWIL1 siRNA in the ex vivo assay shown in Fig. 3d. Western blotting also showed a significant reduction in the levels of MAP1B, MAP2, and Tau, but not doublecortin (DCX) after transfection with PIWIL1 siRNA in cultured cortical neurons (Fig. 4c and d) (Note: Tau image was obtained from the same experiment as that in Fig. 6b).


Novel function of PIWIL1 in neuronal polarization and migration via regulation of microtubule-associated proteins.

Zhao PP, Yao MJ, Chang SY, Gou LT, Liu MF, Qiu ZL, Yuan XB - Mol Brain (2015)

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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Fig6: 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
Mentions: Considering the well-established roles of microtubule dynamics in neuronal polarization, we next focused on the microtubule-associated proteins which may be a potential target of PIWIL1. As shown in Fig. 4b, we found that in the RNAi-treated group the mRNA levels of several microtubule-associated proteins (MAPs), including MAP1B, MAP2, and Tau decreased significantly. Down-regulation of Tau is consistent with a low immunostaining signal of Tau in cultured newborn neurons that had been electroporated with the PIWIL1 siRNA in the ex vivo assay shown in Fig. 3d. Western blotting also showed a significant reduction in the levels of MAP1B, MAP2, and Tau, but not doublecortin (DCX) after transfection with PIWIL1 siRNA in cultured cortical neurons (Fig. 4c and d) (Note: Tau image was obtained from the same experiment as that in Fig. 6b).

Bottom Line: Furthermore, we found that PIWIL1 unexpectedly regulates the expression of microtubule-associated proteins in cortical neurons.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.

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.

No MeSH data available.


Related in: MedlinePlus