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The PtdIns(3,4)P2 phosphatase INPP4A is a suppressor of excitotoxic neuronal death

Abstract

Phosphorylated derivatives of phosphatidylinositol, collectively referred to as phosphoinositides, occur in the cytoplasmic leaflet of cellular membranes and regulate activities such as vesicle transport, cytoskeletal reorganization and signal transduction1,2. Recent studies have indicated an important role for phosphoinositide metabolism in the aetiology of diseases such as cancer, diabetes, myopathy and inflammation3,4,5. Although the biological functions of the phosphatases that regulate phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) have been well characterized, little is known about the functions of the phosphatases regulating the closely related molecule phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2). Here we show that inositol polyphosphate phosphatase 4A (INPP4A), a PtdIns(3,4)P2 phosphatase, is a suppressor of glutamate excitotoxicity in the central nervous system. Targeted disruption of the Inpp4a gene in mice leads to neurodegeneration in the striatum, the input nucleus of the basal ganglia that has a central role in motor and cognitive behaviours. Notably, Inpp4a-/- mice show severe involuntary movement disorders. In vitro, Inpp4a gene silencing via short hairpin RNA renders cultured primary striatal neurons vulnerable to cell death mediated by N-methyl-d-aspartate-type glutamate receptors (NMDARs). Mechanistically, INPP4A is found at the postsynaptic density and regulates synaptic NMDAR localization and NMDAR-mediated excitatory postsynaptic current. Thus, INPP4A protects neurons from excitotoxic cell death and thereby maintains the functional integrity of the brain. Our study demonstrates that PtdIns(3,4)P2, PtdIns(3,4,5)P3 and the phosphatases acting on them can have distinct regulatory roles, and provides insight into the unique aspects and physiological significance of PtdIns(3,4)P2 metabolism. INPP4A represents, to our knowledge, the first signalling protein with a function in neurons to suppress excitotoxic cell death. The discovery of a direct link between PtdIns(3,4)P2 metabolism and the regulation of neurodegeneration and involuntary movements may aid the development of new approaches for the treatment of neurodegenerative disorders.

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Figure 1: Mortality and involuntary movements of Inpp4a -/- mice.
Figure 2: Neurodegeneration in Inpp4a -/- striatum.
Figure 3: INPP4A suppresses glutamate excitotoxicity.
Figure 4: Increased NMDARs at the synapses in the absence of INPP4A.

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Acknowledgements

We thank R. Shigemoto, T. Iwatsubo, I. Kanazawa, T. Shimizu, H. Ichijo, K. Yamada, Y. Imai, K. Kawamura, Y. Kanaho and T. Itoh for discussions; and H. Takahashi, K. Sasaki, Y. Sugihara, S. Kumagai, A. Kato, C. Horie, M. Nishio, S. Sato, T. Sugawara, R. Nakamura and K. Mizoi for technical support. Anti-Met-enkephalin antibody was provided by T. Kaneko and T. Furuta, and anti-NR2C antibody was provided by B. S. Chen and W. Roche. This work was supported by research grants from the Ministry of Education, Culture, Sports and Technology of Japan (MEXT); the Japan Society for the Promotion of Science (JSPS); the Japan Science and Technology Corporation (JST); the Naito Foundation; and the Toray Science Foundation. H.H., T.T., A.S. and T.S. are supported by the Global Centers of Excellence Program of MEXT. T.T. and T.S. are supported by a Grant-in-Aid for Creative Scientific Research from MEXT.

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Authors and Affiliations

Authors

Contributions

J.S. and T.S. designed the research. J.S. generated the Inpp4A-deficient mice. J.S., S.K., R.I., H.M. and Y.T. performed experiments and collected and analysed data. T.M. carried out the electrophysiological studies. K.T. and H.H. performed the lentiviral reconstitution experiments. S.C. performed the ultrastructural analyses. Y.H., S.T., S.E., K.M., K.A. and M.Y. contributed to the histological analyses and provided technical assistance with the shRNA experiments. M.D. and C.M. contributed to the analyses of weeble mice. T.T., A.S. and T.S. supervised the research. T.S. wrote the paper with the input of J.S., T.M., H.H. and M.Y.

Corresponding authors

Correspondence to Junko Sasaki or Takehiko Sasaki.

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Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-8 with legends. (PDF 701 kb)

Supplementary Movie 1

This movie shows that an Inpp4a-/- mouse in a home cage spontaneously exhibits hyperkinetic involuntary movements. (MOV 2827 kb)

Supplementary Movie 2

This movie shows that an Inpp4a-/- mouse placed in a new environment displays severe involuntary movements. (MOV 883 kb)

Supplementary Movie 3

In this movie we see an Inpp4a-/- mouse exhibiting involuntary movements prior to MK801 administration. (MOV 380 kb)

Supplementary Movie 4

In this movie, which was taken 1 hour after MK801 administration, involuntary movements in the same Inpp4a-/- mouse were ameliorated. (MOV 306 kb)

Supplementary Movie 5

In this movie we see that a mouse bearing the Inpp4a null allele and a weeble allele (Inpp4AΔ744G-/- ) exhibits the same involuntary movements as Inpp4a-/- mice. (MOV 368 kb)

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Sasaki, J., Kofuji, S., Itoh, R. et al. The PtdIns(3,4)P2 phosphatase INPP4A is a suppressor of excitotoxic neuronal death. Nature 465, 497–501 (2010). https://doi.org/10.1038/nature09023

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