TY - JOUR
T1 - Disruption of the psychiatric risk gene Ankyrin 3 enhances microtubule dynamics through GSK3/CRMP2 signaling
AU - Garza, Jacob C.
AU - Qi, Xiaoli
AU - Gjeluci, Klaudio
AU - Leussis, Melanie P.
AU - Basu, Himanish
AU - Reis, Surya A.
AU - Zhao, Wen Ning
AU - Piguel, Nicolas H.
AU - Penzes, Peter
AU - Haggarty, Stephen J.
AU - Martens, Gerard J.
AU - Poelmans, Geert
AU - Petryshen, Tracey L.
N1 - Funding Information:
The authors wish to thank Dr. Vann Bennett for providing the Ank3 mouse model, Dr. Feng Zhang for providing the sgRNA plasmid, and Francisca Meyer and Vivian Eijsink for advising on RNA sequencing data analysis. We thank Richard Bouley in the MGH Program in Membrane Biology (PMB) Microscopy Core for help with the confocal imaging. The Nikon A1R confocal in the PMB Microscopy Core was purchased using an NIH Shared Instrumentation Grant S10 RR031563-0. Additional support for the PMB Core came from the Boston Area Diabetes and Endocrinology Research Center (DK057521) and the MGH Center for the Study of Inflammatory Bowel Disease (DK043351). This research was supported by a Brain & Behavior Research Foundation Independent Investigator award (TLP) and Young Investigator award (JCG), NIH grants R21 MH099760 (TLP), K22 NS094591 (JCG), and R01 MH107182 (PP), the Massachusetts General Hospital Executive Committee on Research (TLP and JCG), and the European Community’s Seventh Framework Programme (FP7/ 2007-2013) under grant agreement n° 278948 (TLP). This report reflects only the authors’ views. The funding sponsors are not liable for any use that may be made of the information contained therein.
Publisher Copyright:
© 2018, The Author(s).
PY - 2018/12/1
Y1 - 2018/12/1
N2 - The ankyrin 3 gene (ANK3) is a well-established risk gene for psychiatric illness, but the mechanisms underlying its pathophysiology remain elusive. We examined the molecular effects of disrupting brain-specific Ank3 isoforms in mouse and neuronal model systems. RNA sequencing of hippocampus from Ank3+/− and Ank3+/+ mice identified altered expression of 282 genes that were enriched for microtubule-related functions. Results were supported by increased expression of microtubule end-binding protein 3 (EB3), an indicator of microtubule dynamics, in Ank3+/− mouse hippocampus. Live-cell imaging of EB3 movement in primary neurons from Ank3+/− mice revealed impaired elongation of microtubules. Using a CRISPR-dCas9-KRAB transcriptional repressor in mouse neuro-2a cells, we determined that repression of brain-specific Ank3 increased EB3 expression, decreased tubulin acetylation, and increased the soluble:polymerized tubulin ratio, indicating enhanced microtubule dynamics. These changes were rescued by inhibition of glycogen synthase kinase 3 (GSK3) with lithium or CHIR99021, a highly selective GSK3 inhibitor. Brain-specific Ank3 repression in neuro-2a cells increased GSK3 activity (reduced inhibitory phosphorylation) and elevated collapsin response mediator protein 2 (CRMP2) phosphorylation, a known GSK3 substrate and microtubule-binding protein. Pharmacological inhibition of CRMP2 activity attenuated the rescue of EB3 expression and tubulin polymerization in Ank3-repressed cells by lithium or CHIR99021, suggesting microtubule instability induced by Ank3 repression is dependent on CRMP2 activity. Taken together, our data indicate that ANK3 functions in neuronal microtubule dynamics through GSK3 and its downstream substrate CRMP2. These findings reveal cellular and molecular mechanisms underlying brain-specific ANK3 disruption that may be related to its role in psychiatric illness.
AB - The ankyrin 3 gene (ANK3) is a well-established risk gene for psychiatric illness, but the mechanisms underlying its pathophysiology remain elusive. We examined the molecular effects of disrupting brain-specific Ank3 isoforms in mouse and neuronal model systems. RNA sequencing of hippocampus from Ank3+/− and Ank3+/+ mice identified altered expression of 282 genes that were enriched for microtubule-related functions. Results were supported by increased expression of microtubule end-binding protein 3 (EB3), an indicator of microtubule dynamics, in Ank3+/− mouse hippocampus. Live-cell imaging of EB3 movement in primary neurons from Ank3+/− mice revealed impaired elongation of microtubules. Using a CRISPR-dCas9-KRAB transcriptional repressor in mouse neuro-2a cells, we determined that repression of brain-specific Ank3 increased EB3 expression, decreased tubulin acetylation, and increased the soluble:polymerized tubulin ratio, indicating enhanced microtubule dynamics. These changes were rescued by inhibition of glycogen synthase kinase 3 (GSK3) with lithium or CHIR99021, a highly selective GSK3 inhibitor. Brain-specific Ank3 repression in neuro-2a cells increased GSK3 activity (reduced inhibitory phosphorylation) and elevated collapsin response mediator protein 2 (CRMP2) phosphorylation, a known GSK3 substrate and microtubule-binding protein. Pharmacological inhibition of CRMP2 activity attenuated the rescue of EB3 expression and tubulin polymerization in Ank3-repressed cells by lithium or CHIR99021, suggesting microtubule instability induced by Ank3 repression is dependent on CRMP2 activity. Taken together, our data indicate that ANK3 functions in neuronal microtubule dynamics through GSK3 and its downstream substrate CRMP2. These findings reveal cellular and molecular mechanisms underlying brain-specific ANK3 disruption that may be related to its role in psychiatric illness.
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U2 - 10.1038/s41398-018-0182-y
DO - 10.1038/s41398-018-0182-y
M3 - Article
C2 - 30046097
AN - SCOPUS:85050651940
VL - 8
JO - Translational Psychiatry
JF - Translational Psychiatry
SN - 2158-3188
IS - 1
M1 - 135
ER -