Makoto Itakura

Kitasato University

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)‐dependent neuronal cell survival and differentiation. The small GTPase ADP‐ribosylation factor 6 (Arf6) is implicated in NGF‐dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Ar...

Synovial inflammation plays a central role in joint destruction and pain in osteoarthritis (OA). The NF-κB pathway plays an important role in the inflammatory process and is activated in OA. A previous study reported that a jietacin derivative (JD), (Z)-2-(8-oxodec-9-yn-1-yl)-1-vinyldi-azene 1-oxide, suppressed the nuclear translocation of NF-κB in...

Synovial inflammation plays a crucial role in the destruction of joints and the experience of pain in osteoarthritis (OA). Emerging evidence suggests that certain antibiotic agents and their derivatives possess anti-inflammatory properties. Medermycin (MED) has been identified as a potent antibiotic, specifically active against Gram-positive bacter...

TAR DNA-binding protein 43 kDa (TDP-43), a nuclear protein, plays an important role in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS). The long-disordered C-terminal region (CTR) of TDP-43 is known to be aggregation-prone and a hotspot for ALS mutations, so elucidation of the physiological function of CTR will provide insights in...

Purpose: Carpal tunnel syndrome (CTS) constitutes a typical complication of patients receiving long-term hemodialysis (HD). However, the mechanisms underlying the manifestation of CTS in patients receiving long-term hemodialysis remain to be studied. We investigated collagen gene expression in the subsynovial connective tissue (SSCT) of patients un...

Expression of the apelin receptor, APJ, in skeletal muscle (SM) is known to decrease with age, but the underlying mechanism remains unclear. Increased tumor necrosis factor (TNF)-α levels are observed in SM with age and are associated with muscle atrophy. To investigate the possible interconnection between TNF-α elevation and APJ reduction with agi...

The discovery of bioactive peptides is an important research target that enables the elucidation of the pathophysiology of human diseases and provides seeds for drug discovery. Using a large number of native peptides previously identified using plasma peptidomics technology, we sequentially synthesized selected sequences and subjected them to funct...

Introduction Studies have identified the presence of M1 and M2 macrophages (Mϕ) in injured intervertebral discs (IVDs). However, the origin and polarization-regulatory factor of M2 Mϕ are not fully understood. TGF-β is a regulatory factor for M2 polarization in several tissues. Here, we investigated the source of M2 Mϕ and the role of TGF-β on M2 p...

Dopaminergic neurotransmission via dopamine D1 receptors (D1Rs) is considered to play an important role not only in reward-based learning but also in aversive learning. The contextual and auditory cued fear conditioning tests involve the processing of classical fear conditioning and evaluates aversive learning memory. It is possible to evaluate ave...

Recent mass spectrometry (MS)-based techniques enable deep proteome coverage with relative quantitative analysis, resulting in increased identification of very weak signals accompanied by increased data size of liquid chromatography (LC)–MS/MS spectra. However, the identification of weak signals using an assignment strategy with poorer performance...

TAR DNA-binding protein 43 kDa (TDP-43), a nuclear protein, plays an important role in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS). TDP-43 aggregation and translocation out of the nucleus are crucial factors in ALS. TDP-43 aggregation results from its resistance to degradation, to which the long-disordered C-terminal region (C...

Recent Mass spectrometry (MS)-based techniques enable deep proteome coverage with relative quantitative analysis, resulting in increased identification of very weak signals accompanied by increased data size of liquid chromatography (LC)–MS/MS spectra. However, the identification of weak signals using an assignment strategy with poorer performance...

Background Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). Following disc injury, nerve growth factor (NGF) concentrations rise in IVDs, and anti-NGF therapy has been shown to attenuate LBP in humans. Increased levels of tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) in degenerative IVDs and...

Living organisms contain a variety of endogenous peptides that function as significant regulators of many biological processes. Endogenous peptides are typically analyzed using liquid chromatography–mass spectrometry (LC-MS). However, due to the low efficiency of peptide extraction and low abundance of peptides in a single animal, LC-MS–based pepti...

Shotgun proteomics is a powerful method for comprehensively identifying and quantifying tryptic peptides, but it is difficult to analyze proteolytic events. One-dimensional gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) enables the separation of proteolytic fragments using SDS-PAGE followed by identification using LC-MS/MS. GeL...

GABA is synthesized by glutamate decarboxylase (GAD), which has two isoforms, namely, GAD65 and GAD67, encoded by the Gad2 and Gad1 genes, respectively. GAD65‐deficient (Gad2−/−) mice exhibit a reduction in brain GABA content after 1 month of age and show spontaneous seizures in adulthood. Approximately 25% of Gad2−/− mice died by 6 months of age....

Synapse loss occurs early in Alzheimer’s disease (AD) patients and animal models. Alterations at synaptic level are a major morphological correlate of the memory deficits and related symptoms of AD. Given the predominant roles of synaptic AMPA receptors (AMPARs) in excitatory synaptic transmission in the brain, changes in their dynamic regulation a...

BRAG2/Iqsec1 is a guanine nucleotide exchange factor (GEF) for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes. BRAG2 regulates Arf6-dependent endocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) through the direct interaction during...

Acute brain slice preparation is a powerful experimental model for investigating the characteristics of synaptic function in the brain. Although brain tissue is usually cut at ice-cold temperature (CT) to facilitate slicing and avoid neuronal damage, exposure to CT causes molecular and architectural changes of synapses. To address these issues, we...

Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulatio...

Acute brain slice preparation is a powerful experimental model for investigating the characteristics of synaptic function in the brain. Although brain tissue is usually cut at ice-cold temperature (CT) to facilitate slicing and avoid neuronal damage, exposure to CT causes molecular and architectural changes of synapses. To address these issues, we...

Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. He...

Background: Previous studies suggest the presence of an association of vascular endothelial growth factor (VEGF) with osteoarthritis (OA) severity and pain in patients with knee OA. VEGF expression in human synovial fibroblasts (SFs) is induced by transforming growth factor-beta (TGFβ). However, the signaling pathway governing TGFβ-mediated regula...

ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates various neuronal events including formation of the axon, dendrites and dendritic spines, and synaptic plasticity through actin cytoskeleton remodeling and endosomal trafficking. EFA6C, also known as Psd2, is a guanine nucleotide exchange factor for Arf6 that is preferentially express...

Background Nerve growth factor (NGF) contributes to pain in knee osteoarthritis (KOA) patients. Transforming growth factor-beta (TGF-β) stimulates NGF expression in chondrocytes from KOA patients. However, the correlation between synovial TGF-β and NGF levels has not been sufficiently studied in human KOA patients. Further, the mechanism governing...

Dysfunctional mitochondria are observed in β-cells of diabetic patients, which are eventually removed by autophagy. VAMP7, a vesicular SNARE protein, regulates autophagosome formation to maintain mitochondrial homeostasis and control insulin secretion in pancreatic β-cells. However, its molecular mechanism is largely unknown. Here, we investigated...

SNAP-23 is a plasma membrane-localized SNARE protein involved in Fc receptor (FcR)-mediated phagocytosis. However, the regulatory mechanism underlying its function remains elusive. Using phosphorylation specific-antibodies, SNAP-23 was found to be phosphorylated at Ser95 in macrophages. To understand the role of this phosphorylation, we established...

The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high...

The green fluorescence emission of 9-aminoanthracence (9AA) was maintained by controlling the oxidation of 9AA with oxygen in the solid state and in solution. The solid-state fluorescence of 9AA was maintained for a longer time when lauric acid was used because the equilibrium between 9AA and 9-anthrylammonium salt (9AAH + ) inclines toward the rig...

Background Recent studies have suggested that the tumor necrosis factor-α (TNF-α) pathway is a potential target for the management of osteoarthritis (OA). Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is essential in several cytokine-mediated cascades, including the TNF-α, interleukin-1 (IL-1), and TGF-β pathways. The role of TAK1 in...

Objective: ClC-5 channel is co-localized with the V-ATPase in subapical endosomes of renal proximal tubule and α-intercalated cells. ClC-5 may play a crucial role in regulating both endocytosis and sorting of the acid transporters. This study aimed to elucidate the involvement of ClC-5 in urinary acidification in response to chronic acid loading vi...

A mouse model of epilepsy was generated by inducing status epilepticus (SE) for either 1.5 or 4.5h with pilocarpine to study anxiety-related behaviors, changes in the electroencephalogram of the cerebral cortex and hippocampus, and expression of hippocampal proteins. The viability and rate of success of SE induction were high in C57BL/6N mice but n...

VAMP7 is a SNARE protein that mediates specific membrane fusions in intracellular trafficking and was recently reported to regulate autophagosome formation. However, its function in pancreatic β-cells is largely unknown. To elucidate the physiological role of VAMP7 in β-cells, we generated pancreatic β-cell-specific VAMP7 knockout (Vamp7(flox/Y);Cr...

SNAP-25 is a neurotransmitter vesicular docking protein which has been associated with brain disorders such as attention deficit hyperactivity disorder, bipolar disorder and schizophrenia. In this project, we were interested if clinical factors are associated with differential SNAP-25 expression. We examined the SNAP-25 isoform mRNA and protein lev...

Nitrated guanine nucleotide 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) generated by reactive oxygen/nitrogen species causes protein S-guanylation. However, the mechanism of 8-nitro-cGMP formation and its protein targets in the normal brain have not been identified. Here, we investigated 8-nitro-cGMP generation and protein S-guanylat...

Our open-label pilot study showed that supplementation with docosahexaenoic acid (DHA) increased serum brain-derived neurotrophic factor (BDNF) levels and that there might be an association between changes in serum BDNF levels and reduced psychological distress. Animal research has indicated that a DHA-enriched diet increases BDNF in the brain. In...

Long-term potentiation (LTP) is the key cellular mechanism for physiological learning and pathological chronic pain. Postsynaptic accumulation of AMPA receptor (AMPAR) GluA1 plays an important role for injury-related cortical LTP. However, there is no direct evidence for postsynaptic GluA1 insertion or accumulation after peripheral injury. Here we...

Major histocompatibility complex class I (MHCI) molecules were recently identified as novel regulators of synaptic plasticity. These molecules are expressed in various brain areas, especially in regions undergoing activity-dependent synaptic plasticity, but their role in the nucleus accumbens (NAc) is unknown. In this study, we investigated the eff...

Transmembrane α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate ( AMPA ) receptor regulatory proteins ( TARP s) are auxiliary subunits that regulate AMPA receptor trafficking to the plasma membrane and localization to postsynaptic sites. The classical TARP family consists of four members: stargazin/γ‐2, γ‐3, γ‐4 and γ‐8. The TARP γ‐8 isoform, which...

Brain-derived neurotrophic factor (BDNF) has been studied for one of candidate proteins for brain disorder. Its detection has been performed with Enzyme-Linked Immunosorbent Assay (ELISA) at present. However, developments of more rapid BDNF detection system using a small amount blood sample has been demanded and it is important to find the anti-BDN...

Synaptosomal-associated protein of 25kDa (SNAP-25), a t-SNARE protein, plays a crucial role in neurotransmitter release by exocytosis. Protein kinase C phosphorylates SNAP-25 at Ser(187), however the physiological significance of this phosphorylation event in brain function remains unclear. In the present study, we found that SNAP-25 phosphorylatio...

Calnexin is a molecular chaperone that resides in the endoplasmic reticulum and participates in the folding and assembly of nascent proteins. In the present study, calnexin was found in both synaptic and non-synaptic membrane components of rat brain tissue. Immunohistochemical staining of mouse hippocampal sections revealed the presence of calnexin...

Synaptosomal-associated protein 25 (SNAP-25) plays an essential role in exocytotic neurotransmitter release as a t-SNARE protein. SNAP-25 is phosphorylated at Ser(187) in a protein kinase C (PKC)-dependent manner, but the mechanism for dephosphorylation has yet to be clarified. We investigated SNAP-25 dephosphorylation by comparing it to growth ass...

Pathological examination of dementia with Lewy bodies patients identified the presence of abnormal α-synuclein (αSyn) aggregates in the presynaptic terminals. αSyn is involved in the regulation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Importantly, αSyn-transgenic mouse and postmortem examination of p...

Ipsilateral and contralateral hippocampal CA3-CA1 and CA2-CA1 projections were investigated in adult male Long-Evans rats by retrograde tracing. Injection of the retrograde tracer cholera toxin subunit B in the strata oriens and radiatum of dorsal CA1 resulted in labeling of predominantly pyramidal cells in ipsilateral and contralateral CA3 and CA2...

The Ca2+-dependent activator protein for secretion (CAPS) family consists of two members (CAPS1 and CAPS2) and regulates the exocytosis of catecholamine-containing or neuropeptide-containing dense-core vesicles (DCVs) at secretion sites such as nerve terminals. A large fraction of CAPS1, however, is localized in the cell soma, and we have recently...

Soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-25 is a neuronal SNARE protein essential for neurotransmitter release from presynaptic terminals. Three palmitoylated SNAP-25 family proteins: SNAP-25a, SNAP-25b, and SNAP-23, are expressed in the brain, but little is known about their distributions and functions. In the present st...

Astrocytes release various bioactive substances via Ca(2+) - and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent exocytosis; however the regulatory mechanisms of glial exocytosis are still poorly understood. In the present study, we investigated the effect of protein kinase C (PKC) on exocytosis in glial cell...

Ca(2+)-dependent activator protein for secretion (CAPS) regulates exocytosis of catecholamine- or neuropeptide-containing dense-core vesicles (DCVs) at secretion sites, such as nerve terminals. However, large amounts of CAPS protein are localized in the cell soma, and the role of somal CAPS protein remains unclear. The present study shows that soma...

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors require auxiliary subunits termed transmembrane AMPA receptor regulatory proteins (TARPs), which promote receptor trafficking to the cell surface and synapses and modulate channel pharmacology and gating. Of six TARPs, gamma-2 and gamma-7 are the two major...

Left-right asymmetry of the brain has been studied mostly through psychological examination and functional imaging in primates, leaving its molecular and synaptic aspects largely unaddressed. Here, we show that hippocampal CA1 pyramidal cell synapses differ in size, shape, and glutamate receptor expression depending on the laterality of presynaptic...

Ionotropic glutamate receptors play important roles in spinal processing of nociceptive sensory signals and induction of central sensitization in chronic pain. Here we applied highly sensitive freeze-fracture replica labeling to laminae I-II of the spinal dorsal horn of rats and investigated the numbers, densities, and colocalization of AMPA- and N...

Astrocyte secrete many kinds of bioactive substances including growth factors and cytokines. Recent accumulating evidences showed that astrocyte also release neurotransmitters and modulators such as glutamate, ATP and D-serine. At least, some part of these releases are mediated by exocytosis in SNARE- and Ca2+-dependent manners. These functions may...

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter re...

Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1)....

Evidence that glutamate and ATP release from astrocytes can occur via gap junction hemichannels (GJHCs) is accumulating. However, the GJHC is still only one possible release mechanism and has not been detected in some studies, although this may be because the levels were below those detectable by the system used. Because of these conflicting result...

Cyclin-dependent kinase 5 (Cdk5)-p35 is downregulated in cultured neurons by N-methyl-D-aspartate (NMDA) via the proteasomal degradation of p35. However, it is not known where in neurons this downregulation occurs or the physiologic meaning of the reaction. We show the enrichment of Cdk5 and p35 in the postsynaptic density and the NMDA-induced degr...

Transmembrane AMPA receptor regulatory proteins (TARPs), including stargazin/gamma-2, are associated with AMPA receptors and participate in their surface delivery and anchoring at the postsynaptic membrane. TARPs may also act as a positive modulator of the AMPA receptor ion channel function; however, little is known about other TARP members except...

The Ca(2+)-dependent activator protein for secretion (CAPS/Cadps) family consists of two members, CAPS1 and CAPS2, and plays an important role in secretory granule exocytosis. It has been shown that CAPS1 regulates catecholamine release from neuroendocrine cells, whereas CAPS2 is involved in the release of two neurotrophins, brain-derived neurotrop...

We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent sign...

Recent studies have indicated that various growth factors are involved in synaptic functions; however, the precise mechanisms remain unclear. In order to elucidate the molecular mechanisms of the growth factor-mediated regulation of presynaptic functions, the effects of epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) on neuro...

Synaptic transmission is conducted by neurotransmitters released from nerve terminals. Neurotransmitter release is regulated both positively and negatively by multiple mechanisms, and its regulation is believed to be one of the important mechanisms of synaptic plasticity underlying learning and memory. Various protein kinases play important roles i...

Neurotrophins play important roles in the differentiation and survival of neurons during development, and in the regulation of synaptic transmission in adult brain. Brief treatment with nerve growth factor (NGF) enhances depolarization and ionomycin-induced dopamine and acetylcholine release from PC12 cells. The enhancing effect appears very quickl...

In order to elucidate the molecular mechanism of phorbol ester-induced potentiation of neurotransmitter release, changes in the subcellular distribution of secretory vesicles were studied in PC12 cells. Dopamine (DA) and acetylcholine containing vesicles were selectively labelled by expressing green fluorescent protein-conjugated vesicular monoamin...

Neuropsin involved in neural plasticity in adult mouse brain is a member of the S1 (clan SA) family of serine proteases and forms characteristic surface loops surrounding the substrate-binding site (Kishi, T., Kato, M., Shimizu, T., Kato, K., Matsumoto, K., Yoshida, S., Shiosaka, S., and Hakoshima, T. (1999)J. Biol. Chem. 274, 4220–4224). Little, h...

Clonal rat pheochromocytoma (PC12) cells have been widely used to study the molecular mechanism of exocytosis. We have isolated variant PC12 subclones with deficiencies in stimulation-secretion coupling, by a single cell recloning, and investigated the defects. PC12-1G2 hardly released dopamine following high-K(+)-induced depolarization, but normal...

It has been proposed that the cortical actin filament networks act as a cortical barrier that must be reorganized to enable docking and fusion of the synaptic vesicles with the plasma membranes. We identified a novel neuron-associated developmentally regulated protein, designated as Nadrin. Expression of Nadrin is restricted to neurons and correlat...

Classical neurotransmitters such as gamma-aminobutyric acid and glutamate are released from synaptic nerve terminals by exocytosis of synaptic vesicles. PC12 cells also have SSVs capable of storing acetylcholine (ACh). A novel method to examine the effect of transient transfection of any gene of interest on the exocytosis of SSVs was developed. The...

Natriuretic peptide receptor-C (NPR-C) has a unique structure consisting of pre-existing covalent homodimers, but it is not known whether each subunit has ligand-binding activity or whether the dimeric structure is necessary for binding activity. To answer this question, a number of C-terminally truncated mutants were designed, subcloned into the m...

Through the analysis of the porcine gene encoding the elastase inhibitor elafin, we demonstrated that there are at least three closely related members of the elafin family, and their genes have arisen by accelerated evolution. A porcine genomic DNA library was screened with a previously cloned human elafin cDNA probe, and several positive clones we...

A large number of proteolytic systems are assembled in our body and their activities are strictly regulated by a delicate balance between the production, activation, and inhibition of the proteinases. Since any disturbance in the finely tuned balance of the systems may trigger aggressive destruction of the tissue integrity and normal physiology, a...

The primary and gene structures and tissue distribution of porcine SPAI-2, a protein that belongs to the WAP protein superfamily and has a sodium-potassium ATPase inhibitory activity, were determined by molecular cloning and Northern analysis. A full-length cDNA clone was isolated from a porcine duodenum cDNA library. The cDNA insert encoded a poly...

Receptors for natriuretic peptide (NP) consist of three subtypes: NP-A, NP-B, and NP-C. Recent studies in cultured aortic cells have suggested a phenotype-related switching of the vascular NP receptor from NP-A to NP-B. To ascertain the biological significance of the phenomenon in vivo, we developed a sensitive and reproducible ribonuclease protect...

Receptors for natriuretic peptide (NP) consist of three subtypes: NP-A, NP-B, and NP-C. Recent studies in cultured aortic cells have suggested a phenotype-related switching of the vascular NP receptor from NP-A to NP-B. To ascertain the biological significance of the phenomenon in vivo, we developed a sensitive and reproducible ribonuclease protect...