Interaction of NAP-22 with brain glutamic acid decarboxylase (GAD)

Maékawa, Shohei; Kobayashi, Yuumi; Odagaki, Sin-Ichi; Makino, M.; Kumanogoh, Haruko; Nakamura, Shun; Morita, Mitsuhiro; Hayashi, Fumio

doi:10.1016/j.neulet.2013.01.030

Cited by 13 publications

(13 citation statements)

References 29 publications

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“…Brain acid‐soluble protein 1, a neuron‐enriched protein localized mainly in synaptic vesicles (Maekawa et al . ), was decreased to 62.4% in the HIP. Septin‐8, which controls the binding of vesicle‐associated membrane protein 2 to synaptophysin and participates in neurotransmitter release (Ito et al .…”

Section: Resultsmentioning

confidence: 80%

Evidence of altered depression and dementia‐related proteins in the brains of young rats after ovariectomy

Fang

Zeng

et al. 2018

Journal of Neurochemistry

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Menopause, a risk factor for brain dysfunction in women, is characterized by neuropsychological symptoms including depression and dementia, which are closely related to alterations in different brain regions after menopause. However, little is known about the variability in pathophysiologic changes associated with menopause in the brain. Here, we observed that menopause in rats induced by bilateral ovariectomy (OVX) showed depressive and dementia-related behaviors along with neuronal loss in the prefrontal cortex (PFC), hippocampus (HIP), hypothalamus (HYP), and amygdala (AMY) by Nissl staining. Meanwhile, by immunohistochemical staining, increased microglia in the HIP and AMY and increased astrocytes in the PFC, HYP, and AMY were shown. Using quantitative proteomics, we identified 146 differentially expressed proteins in the brains of OVX rats, for example, 20 in the PFC, 41 in the HIP, 17 in the HYP, and 79 in the AMY, and performed further detection by western blotting. A link between neuronal loss and apoptosis was suggested, as evidenced by increases in adenylate kinase 2 (AK2), B-cell lymphoma 2 associated X (Bax), cleaved caspase 3, and phosphorylated p53 and decreases in Huntingtin-interacting protein K, hexokinase, and phosphorylated B-cell lymphoma 2 (Bcl-2), and apoptosis might be triggered by endoplasmic reticulum stress (probed by increased glucose-regulated protein 78 (GRP78), cleaved caspase 12, phosphorylated protein kinase R (PKR)-like endoplasmic reticulum kinase, inositol-requiring enzyme-1 and activating transcription factor 6), and mitochondrial dysfunction (probed by increased cytochrome c and cleaved caspase 3 and decreased sideroflexin-1 (SFXN1) and NADH dehydrogenase (ubiquinone) 1 α subcomplex 11 (NDUFA11)). Activation of autophagy was also indicated by increased autophagy-related 7, γ-aminobutyric acid (GABA) receptor-associated protein-like 2, and oxysterol-binding protein-related protein 1 and confirmed by increased microtubule-associated protein light chain 3 (LC3II/I), autophagy-related 5, and Beclin1 in the HIP and AMY. In the AMY, which is important in emotion, higher GABA transporter 3 and lower vesicular glutamate transporter 1 levels indicated an imbalance between excitatory and inhibitory neurotransmission, and the increased calretinin and decreased calbindin levels suggested an adjustment of GABAergic transmission after OVX. In addition, cytoskeletal abnormalities including tau hyperphosphorylation, dysregulated Ca² signals, and glutamic synaptic impairments were observed in the brains of OVX rats. Collectively, our study showed the changes in different brain regions related to depression and dementia during menopause.

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Section: Resultsmentioning

confidence: 80%

Evidence of altered depression and dementia‐related proteins in the brains of young rats after ovariectomy

Fang

Zeng

et al. 2018

Journal of Neurochemistry

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“…In addition to NAP‐22, many proteins were detected in the fractions, and some proteins were enriched in the NAP‐22‐bound beads. These bands were analyzed with liquid chromatography‐tandem mass spectrometry (LC‐MS/MS), and one band was found to contain glutamate decarboxylase (Maekawa et al, ). CaN was also detected through LC‐MS/MS analysis, and Western blotting confirmed the result (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Anti‐CaN antibody used in this study reacted with a single band of molecular mass of 60 kDa on Western blotting with a crude soluble fraction of rat brain. Anti‐NAP‐22 monoclonal antibody used in this study reacted with purified NAP‐22 and a single band in whole‐brain extract of rat brain with Western blotting (Maekawa et al, ). In cell staining, no staining was observed without these primary antibodies.…”

Section: Methodsmentioning

confidence: 99%

“…The immunoprecipitation assay used previously showed several other proteins to be candidates for interacting proteins of NAP‐22 (Maekawa et al, ). The current study identifies a 60‐kDa protein as calcineurin (CaN) that was recovered in the immunoprecipitate from the crude soluble fraction of rat brain by using anti‐NAP‐22 monoclonal antibody‐coupled Sepharose 4B beads on which NAP‐22 fraction from brain DRM fraction was precharged.…”

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confidence: 99%

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Ganglioside contained in the neuronal tissue‐enriched acidic protein of 22 kDa (NAP‐22) fraction prepared from the detergent‐resistant membrane microdomain of rat brain inhibits the phosphatase activity of calcineurin

Kobayashi

Silva

Kumanogoh

et al. 2015

J of Neuroscience Research

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Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant membrane microdomain fraction (DRM). Neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) is one of the major protein components of neuronal DRM. To determine the cellular function of NAP-22, interacting proteins were screened with an immunoprecipitation assay, and calcineurin (CaN) was detected. Further studies with NAP-22 prepared from DRM and CaN expressed in bacteria showed the binding of these proteins and a dose-dependent inhibitory effect of the NAP-22 fraction on the phosphatase activity of CaN. On the other hand, NAP-22 expressed in bacteria showed low binding to CaN and a weak inhibitory effect on phosphatase activity. To solve this discrepancy, identification of a nonprotein component that modulates CaN activity in the DRM-derived NAP-22 fraction was attempted. After lyophilization, a lipid fraction was extracted with chloroform/methanol. The lipid fraction showed an inhibitory effect on CaN without NAP-22, and further fractionation of the extract with thin-layer chromatography showed the presence of several lipid bands having an inhibitory effect on CaN. The mobility of these bands coincided with that of authentic ganglioside (GM1a, GD1a, GD1b, and GT1b), and authentic ganglioside showed an inhibitory effect on CaN. Treatment of lipid with endoglycoceramidase, which degrades ganglioside to glycochain and ceramide, caused a diminution of the inhibitory effect. These results show that DRM-derived NAP-22 binds several lipids, including ganglioside, and that ganglioside inhibits the phosphatase activity of CaN.

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“…Cholesterol distribution and the localization of this protein were hence considered intimately related [10]. In order to elucidate additional functions of NAP-22, a screening of NAP-22-binding proteins was performed using NAP-22-coupled Sepharose beads and several proteins such as an actin binding protein (CapZ␣) [15], synaptojanin [21], and glutamate decarboxylase (GAD) [12] were identified. Other functions of NAP-22 in transcriptional regulation, apoptosis, and ion channel formation were also reported by other groups [3,16,17].…”

Section: Introductionmentioning

confidence: 99%