Disruption of neuronal nitric oxide synthase dimerization contributes to the development of Alzheimer's disease: Involvement of cyclin-dependent kinase 5-mediated phosphorylation of neuronal nitric oxide synthase at Ser(293).
Although previous studies have suggested that neuronal nitric oxide synthase (nNOS)-derived NO has neuroprotective effects on the development of Alzheimer's disease (AD), the underlying molecular mechanisms are not fully elucidated. Here, we investigated whether and how disruption of nNOS dimerization contributes to the development of AD. No differences in synaptic number or expression of synaptic markers, including synaptophysin and postsynaptic density 95, were found in the cortex of 5 × FAD mice, which possess 5 familial AD mutations, at 6 months of age compared with control littermates. nNOS dimerization was disrupted in the 5 × FAD cortex, accompanied by an increase in reactive oxygen species (ROS) production. The subcellular distribution of cyclin-dependent kinase 5 (CDK5) shifted more diffusely toward a cytosolic compartment, but there was no change in total expression. Furthermore, the levels of p25, a CDK5 activator, increased significantly and it colocalized with nNOS in the 5 × FAD cortex. In silico analysis revealed that a new nNOS-specific GSP (glycine-serine-proline) motif was well-conserved across species at nNOS-Ser(293), which is located ahead of the N-terminal hook. This motif was not present in the closely related isoform, endothelial NOS. Motif scan analysis also predicted that CDK5 can phosphorylate nNOS-Ser(293) with a high likelihood. An in vitro phosphorylation assay clearly showed that CDK5/p25 does indeed phosphorylate nNOS-Ser(293). Finally, nNOS-S293D mutant, a phosphomimetic form of nNOS-Ser(293), and nNOS-S293A mutant, a neutral form of nNOS-Ser(293), significantly decreased nNOS dimerization and NO production. Taken together, our results demonstrate that nNOS dimers are disrupted in the 5 × FAD cortex, and nNOS-Ser(293), a potential site of CDK5 phosphorylation, may be involved in the decrease in nNOS dimerization and NO production, and the development of AD.
Department of Neurology, Konkuk University Medical Center and Department of Neuroscience, Center for Geriatric Neuroscience Research, Institute of Biomedical Science and Technology, Konkuk University School of Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, South Korea.
Kyoung Ja
KJ
Kwon
Department of Molecular Medicine, Ewha Womans University Medical School, 911-1, Mok-6-dong, Yangchun-gu, Seoul 158-710, South Korea.
Jung-Hyun
JH
Park
Department of Molecular Medicine, Ewha Womans University Medical School, 911-1, Mok-6-dong, Yangchun-gu, Seoul 158-710, South Korea.
Inho
I
Jo
Department of Internal Medicine, Konkuk University School of Medicine, 120-1 Neungdong-ro, Gwangjin-gu, Seoul 143-729, South Korea.
Kee-Ho
KH
Song
Department of Biological Sciences, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, South Korea.
Jung-Soo
JS
Han
Department of Neurology, Konkuk University Medical Center and Department of Neuroscience, Center for Geriatric Neuroscience Research, Institute of Biomedical Science and Technology, Konkuk University School of Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, South Korea.
Seung Hwa
SH
Park
Department of Neurology, Konkuk University Medical Center and Department of Neuroscience, Center for Geriatric Neuroscience Research, Institute of Biomedical Science and Technology, Konkuk University School of Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, South Korea. Electronic address: alzdoc@kuh.ac.kr.
Seol-Heui
SH
Han
Department of Pharmacology, School of Medicine, Eulji University, 77 Gyeryong-ro 771 Beon-gil, Jung-gu, Daejeon 301-746, South Korea. Electronic address: biohyong@hanmail.net.
Du-Hyong
DH
Cho
D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism / Q000473:pathology; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D052248:Nitric Oxide Synthase Type I / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization / Q000502:physiology*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*
D017382:Reactive Oxygen Species; D012694:Serine; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; D051360:Cyclin-Dependent Kinase 5; C495906:Cdk5 protein, mouse
Alzheimer’s disease; Cyclin-dependent kinase 5; Dimerization; Neuronal nitric oxide synthase; Phosphorylation; p25