Progranulin deficiency induces overactivation of WNT5A expression via TNF-α/NF-κB pathway in peripheral cells from frontotemporal dementia-linked granulin mutation carriers

Progranulin deficiency induces overactivation of WNT5A expression via TNF-α/NF-κB pathway in peripheral cells from frontotemporal dementia-linked granulin mutation carriers

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J Psychiatry Neurosci 2016;41(4):225-239

Carolina Alquézar, PhD; Ana de la Encarnación, MSc; Fermín Moreno, MD, PhD; Adolfo López de Munain, MD, PhD; Ángeles Martín-Requero, PhD

Abstract

Background: Loss-of-function progranulin gene (GRN) mutations have been identified as the major cause of frontotemporal lobar degeneration with transactive response (TAR) DNA-binding protein 43 (TDP-43) pathology (frontotemporal lobar degeneration [FTLD]-TDP); however, little is known about the association between progranulin (PGRN) deficiency and neuronal loss in individuals with FTLDTDP. Previously we reported enhanced proliferative activity associated with the activation of WNT5A/CDK6/pRb signalling in PGRN-deficient cells. The objective of this work was to elucidate the association between PGRN deficiency, WNT5A signalling and cell proliferation in immortalized lymphoblasts from carriers of the c.709–1G > A GRN mutation (asymptomatic and FTLD-TDP).

Methods: We assessed cell proliferation in carriers of the c.709–1G > A GRN gene mutation and controls without GRN mutation and without sign of neurologic degeneration by cell counting or using an MTT assay. We used a luciferase assay to measure the nuclear factor-κ (NF-κ) activity. We evaluated messenger RNA levels using quantitative real-time polymerase chain reaction and protein levels by immunoblotting. Co-immunoprecipitation was used to analyze the interaction between PGRN and its receptors.

Results: We enrolled 19 carriers of the GRN gene mutation and 10 controls in this study. The PGRN-deficient cells showed increased expression of WNT5A due to NF-κB signalling overactivation. We observed a competition between PGRN and tumour necrosis factor-α (TNF-α) for binding both TNF receptors (TNFR) I and II. Blocking NF-κB signalling using wedelolactone or specific antibodies against TNFRs inhibited WNT5A overexpression and proliferation of PGRN-deficient cells. Conversely, the activation of NF-κB signalling by TNF-α increased WNT5A-dependent proliferation of control cells.

Limitations: All cell lines were derived from individuals harboring the same splicing GRN mutation. Nevertheless, most of the known GRN mutations lead to haploinsufficiency of the protein.

Conclusion: Our results revealed an important role of NF-κB signalling in PGRN-associated FTLD-TDP and confirm that PGRN can bind to TNF-a receptors regulating the expression of WNT5A, suggesting novel targets for treatment of FTLD-TDP linked to GRN mutations.


Submitted Apr. 16, 2015; Revised July 15, 2015; Accepted Aug. 20, 2015; Early-released Dec. 1, 2015

Acknowledgements: The authors thank Drs. F. Aillet and M.S. Rodríguez for helpful criticism and for their generous gift of the NF-κB luciferase reporter plasmid.

Affiliations: From the Department of Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas (CSIC) Madrid (Alquézar, de la Encarnación, Martín-Requero); the Neuroscience Area-Instituto Biodonostia, Hospital Universitario Donostia, San Sebastian, Spain (Moreno, López de Munain); the Department of Neurology, Hospital Donostia, San Sebastian, Spain (Moreno); the Department of Neurosciences, University of Basque Country, San Sebastian, Spain (López de Munain); the CIBER de Enfermedades Neurodegenerativas (CIBERNED) (López de Munain) and the CIBER de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, Spain (Martín-Requero).

Funding: This work has been supported by grants from Ministerio de Economía y Competitividad (SAF2011-28603) and Fundación Ramón Areces to Á. Martín-Requero. A. López de Munain received research support from the Ilundain Foundation.

Competing interests: None declared.

Contributors: C. Alquézar and Á. Martín-Requero designed the study. C. Alquézar and A. de la Encarnación acquired the data, which all authors analyzed. All authors wrote and reviewed the article and approved the final version for publication.

DOI: 10.1503/jpn.150131

Correspondence to: Á. Martín-Requero, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain; amrequero@cib.csic.es