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TRPM7 Regulates Axonal Outgrowth and Maturation of Primary Hippocampal Neurons

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Abstract

Transient receptor potential melastatin 7 (TRPM7) is a calcium-permeable divalent cation channel and mediates neuronal cell death under ischemic stresses. In this study, we investigated the contribution of TRPM7 to neuronal development in mouse primary hippocampal neurons. We demonstrated that TRPM7 channels are highly expressed in the tips of the growth cone. Either knockdown of TRPM7 with target-specific shRNA or blocking channel conductance by a specific blocker waixenicin A enhanced axonal outgrowth in culture. Blocking TRPM7 activity by waixenicin A reduced calcium influx and accelerated the polarization of the hippocampal neurons as characterized by the development of distinct axons and dendrites. Furthermore, TRPM7 coprecipitated and colocalized with F-actin and α-actinin-1 at the growth cone. We conclude that calcium influx through TRPM7 inhibits axonal outgrowth and maturation by regulating the F-actin and α-actinin-1 protein complex. Inhibition of TRPM7 channel promotes axonal outgrowth, suggesting its therapeutic potential in neurodegenerative disorders.

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Abbreviations

TRPM7 channel:

Transient receptor potential melastatin 7 channel

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Acknowledgments

This work was supported by the following grants: NIH NIGMS P01 (GM078195) to AF, Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants to ZPF (RGPIN 249962) and to HSS (RGPIN 402733), a Canada Foundation for Innovation (CFI #29066) Leader of Opportunity Fund and Ontario Research Fund (ORF) to HSS, a Canadian Institute of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarship to ET, Ontario Graduate Scholarships to CB (OGS-MSc) and to AB (OGS-PhD), and a New Investigator Award from the Heart and Stroke Foundation of Canada (HSFC) to ZPF.

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Authors and Affiliations

  1. Department of Surgery, Faculty of Medicine, University of Toronto, 1132 Medical Sciences Building, 1 King’s College Circle, Toronto, ON, M5S 1A8, Canada

    Ekaterina Turlova, Christine Y. J. Bae, Wenliang Chen & Hong-Shuo Sun

  2. Department of Physiology, Faculty of Medicine, University of Toronto, 3306 Medical Sciences Building, 1 King’s College Circle, Toronto, ON, M5S 1A8, Canada

    Ekaterina Turlova, Christine Y. J. Bae, Marielle Deurloo, Wenliang Chen, Andrew Barszczyk, Zhong-Ping Feng & Hong-Shuo Sun

  3. Department of Pharmacology & Toxicology, Faculty of Medicine, University of Toronto, 1 King’s College Circle, Toronto, ON, M5S 1A8, Canada

    Hong-Shuo Sun

  4. Institute of Medical Science, Faculty of Medicine, University of Toronto, 1 King’s College Circle, Toronto, ON, M5S 1A8, Canada

    Hong-Shuo Sun

  5. College of Natural and Computational Sciences, Hawaii Pacific University, Kaneohe, HI, 96744, USA

    F. David Horgen

  6. Center for Biomedical Research, The Queen’s Medical Center, Honolulu, HI, 96720, USA

    Andrea Fleig

  7. University of Hawaii Cancer Center and John A. Burns School of Medicine, Honolulu, HI, 96720, USA

    Andrea Fleig

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  1. Ekaterina Turlova
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Correspondence to Zhong-Ping Feng or Hong-Shuo Sun.

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Supplementary Figure 1

Confirmation of AAV-1 vector efficiency. A) AAV-1 vectors infected neurons with near 100% efficiency, determined by EGFP expression. Comparing between the number of neurons in top image (indicated by red dots, n = 27) and EGFP fluorescent neurons (n = 26) in the bottom image, 96.3% of the neurons were infected. B) Western blot showing a single protein band for hippocampal (HPC) and prefrontal cortex (PFC) regions of mouse and rat brain tissues labeled by the TRPM7 antibody used in C. C) Representative confocal immunofluorescence images obtained from E17.5 mouse hippocampal neurons on DIV6 treated with AAV-1 viruses containing eGFP only (n = 28), scrambled shRNA (n = 26), or TRPM7 shRNA (n = 43). eGFP (green), NeuN (red), and TRPM7 (blue) are shown. D) Summary of the relative expression levels of TRPM7 against NeuN. The graph is expressed as mean ± SEM (*** p <0.001). (GIF 213 kb)

High resolution image (TIFF 13860 kb)

Supplementary Figure 2

Neurite analysis of TRPM7 knockdown neurons. A) A schematic representation of primary, secondary and tertiary neurites. B) Number of primary, secondary and tertiary of DIV4, C) DIV6 and D) DIV8 neurons in three different treatment groups. For groups at DIV4: eGFP only n = 42, scrambled shRNA n = 43, TRPM7 shRNA n = 48; for groups at DIV6: eGFP only n = 26, scrambled shRNA n = 40, TRPM7 shRNA n = 35; for groups at DIV8: eGFP only n = 25, scrambled shRNA n = 20, TRPM7 shRNA n = 23. Data is shown as mean ± SEM. Statistical analysis: One-way ANOVA with Bonferroni post-hoc. (GIF 74 kb)

High resolution image (TIFF 12340 kb)

Supplementary Figure 3

Quantitative analysis of neuronal transition between second and third developmental stages. A) Formula for calculating the difference between the length of longest neurite and the average length of the remaining neurites. B) Average length difference of DIV2 and DIV3 neurons in different treatment groups. C) Average length difference of stage 2 and stage 3 neurons in different treatment groups at DIV2 and DIV3. Data is presented as mean ± SEM. Statistical analysis: one-way ANOVA with Bonferroni post-hoc (*p < 0.05; ***p < 0.001). D) Distribution of neurons in stage 2 and stage 3 in different treatment groups at DIV2 and DIV3, based on the assumption that the length difference for stage 3 is ≥ 0.67. Chi-square: DIV2 untreated control vs. waixenicin A P = 0.0015; DIV3 untreated control vs. waixenicin A P = 0.0046. For groups at DIV2: untreated control n = 73; vehicle control n = 68; waixenicin A n = 69. For groups at DIV3: untreated control n = 63; vehicle control n = 62; waixenicin A n = 62. (GIF 119 kb)

High resolution image (TIFF 18104 kb)

Supplementary Table 1

Summary of Pearson correlation coefficient values for the colocalization analysis. Summary of Rrs in different neuronal structures and the % change in Rr in the between the control and waixenicin A-treated neurons. (GIF 66 kb)

High resolution image (TIFF 6759 kb)

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Turlova, E., Bae, C.Y.J., Deurloo, M. et al. TRPM7 Regulates Axonal Outgrowth and Maturation of Primary Hippocampal Neurons. Mol Neurobiol 53, 595–610 (2016). https://doi.org/10.1007/s12035-014-9032-y

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