Toshihide Kimura

CRMP-2 binds to tubulin heterodimers to promote microtubule assembly

Regulated increase in the formation of microtubule arrays is thought to be important for axonal growth. Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33, mutations in which result in abnormal axon termination. We recently demonstrated that CRMP-2 is critical for axonal differentiation. Here, we identify two activities of CRMP-2: tubulin-heterodimer binding and the promotion of microtubule assembly. CRMP-2 bound tubulin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tubulin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtubule assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulates axonal growth and branching as a partner of the tubulin heterodimer, in a different fashion from traditional MAPs.

Anterograde transport of TrkB in axons is mediated by direct interaction with Slp1 and Rab27.

The neurotrophin receptors TrkA, TrkB, and TrkC are localized at the surface of the axon terminus and transmit key signals from brain-derived neurotrophic factor (BDNF) for diverse effects on neuronal survival, differentiation, and axon formation. Trk receptors are sorted into axons via the anterograde transport of vesicles and are then inserted into axonal plasma membranes. However, the transport mechanism remains largely unknown. Here, we show that the Slp1/Rab27B/CRMP-2 complex directly links TrkB to Kinesin-1, and that this association is required for the anterograde transport of TrkB-containing vesicles. The cytoplasmic tail of TrkB binds to Slp1 in a Rab27B-dependent manner, and CRMP-2 connects Slp1 to Kinesin-1. Knockdown of these molecules by siRNA reduces the anterograde transport and membrane targeting of TrkB, thereby inhibiting BDNF-induced ERK1/2 phosphorylation in axons. Our data reveal a molecular mechanism for the selective anterograde transport of TrkB in axons and show how the transport is coupled to BDNF signaling.

Axon specification in hippocampal neurons.

Neurons are the most highly polarized cells, comprised of two structurally and functionally distinct parts, axons and dendrites. This asymmetry enables a vectorial flow of signaling within neurons. One of the most fundamental questions still to be answered in neuroscience is how these two specialized processes initially develop. The first manifestation of polarization occurs when one of the immature neurites acquires axonal characteristics. We review recent advances that have highlighted the involvement of several cellular events in the initial formation of the axon, including membrane traffic and cytoskeletal rearrangement. We then discuss the molecular mechanisms underlying axon formation, focusing on the Rho family small GTPases and an axon-inducing neuronal protein, CRMP-2.

Protection of pancreatic -cells by exendin-4 may involve the reduction of endoplasmic reticulum stress; in vivo and in vitro studies

The aim of this study was to investigate the in vivo and in vitro effectsofexendin-4,apotentglucagon-likepeptide1agonist,on theprotectionofthepancreaticb-cellsagainsttheircelldeath.In in vivo experiments, we used b-cell-specific calmodulinoverexpressing mice where massive apoptosis takes place in their b-cells, and we examined the effects of chronic treatment with exendin-4. Chronic and s.c. administration of exendin-4 reduced hyperglycemia. The treatment caused significant increases of the insulin contents of the pancreas and islets, and retained the insulin-positive area. Dispersed transgenic islet cells lived only shortly, and several endoplasmic reticulum (ER) stress-related molecules such as immunoglobulin-binding protein (Bip), inositol-requiring enzyme-1 a ,X -box-binding protein-1 (XBP-1), RNA-activated protein kinase-like endoplasmic reticulum kinase, activating transcription factor-4, and C/EBP-homologous protein (CHOP) were more expressed in the transgenic islets. We also found that the spliced form of XBP-1, a marker of ER stress, was also increased in b-cellspecific calmodulin-overexpressing transgenic islets. In the quantitative real-time PCR analyses, the expression levels of Bip and CHOP were reduced in the islets from the transgenic mice treated with exendin-4. These findings suggest that excess ofERstressoccursinthetransgenicb-cells,andthesuppression of ER stress and resultant protection against cell death may be involved in the anti-diabetic effects of exendin-4.

Glucose-induced production of hydrogen sulfide may protect the pancreatic beta-cells from apoptotic cell death by high glucose

We examined the expression of the major H2S‐producing enzymes, cystathionine‐β‐synthase (CBS) and cystathionine‐γ‐lyase (CSE). CBS was ubiquitously distributed in the mouse pancreas, but CSE was found only in the exocrine. Freshly isolated islets expressed CBS, while CSE was faint. However, high glucose increased the CSE expression in the beta‐cells. l‐Cysteine or NaHS suppressed islet cell apoptosis with high glucose, and increased glutathione content in MIN6 beta‐cells. Pretreatment with l‐cysteine improved the secretory responsiveness following stimulation with glucose. The CSE inhibitor dl‐propargylglycine antagonized these l‐cysteine effects. We suggest H2 S may function as an ‘intrinsic brake’ which protects beta‐cells from glucotoxicity.

Hydrogen sulphide protects mouse pancreatic β-cells from cell death induced by oxidative stress, but not by endoplasmic reticulum stress

BACKGROUND AND PURPOSE Hydrogen sulphide (H2S), a potentially toxic gas, is also involved in the neuroprotection, neuromodulation, cardioprotection, vasodilatation and the regulation of inflammatory response and insulin secretion. We have recently reported that H2S suppresses pancreatic β‐cell apoptosis induced by long‐term exposure to high glucose. Here we examined the protective effects of sodium hydrosulphide (NaHS), an H2S donor, on various types of β‐cell damage.

Distribution of hydrogen sulfide (H2S)-producing enzymes and the roles of the H2S donor sodium hydrosulfide in diabetic nephropathy

BackgroundHydrogen sulfide (H2S) has recently been found to play beneficial roles in ameliorating several diseases, including hypertension, atherosclerosis and cardiac/renal ischemia–reperfusion injuries. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), the main enzymes in the transsulfuration pathway, catalyze H2S production in mammalian tissues. However, the distributions and precise roles of these enzymes in the kidney have not yet been identified.MethodsThe present study examined the localization of both enzymes in the normal kidney and the effect of the H2S donor sodium hydrosulfide (NaHS) in the renal peritubular capillary (PTC) under conditions of diabetic nephropathy, using pancreatic β-cell-specific calmodulin-overexpressing transgenic mice as a model of diabetes.ResultsIn the normal kidney, we detected expression of both CBS and CSE in the brush border and cytoplasm of the proximal tubules, but not in the glomeruli, distal tubules and vascular endothelial cells of renal PTCs. Administration of NaHS increased PTC diameter and blood flow. We further evaluated whether biosynthesis of H2S was altered in a spontaneous diabetic model that developed renal lesions similar to human diabetic nephropathy. CSE expression was markedly reduced under diabetic conditions, whereas CBS expression was unaffected. Progressive diabetic nephropathy showed vasoconstriction and a loss of blood flow in PTCs that was ameliorated by NaHS treatment.ConclusionThese findings suggest that CSE expression in the proximal tubules may also regulate tubulointerstitial microcirculation via H2S production. H2S may represent a target of treatment to prevent progression of ischemic injury in diabetic nephropathy.

The GDP-dependent Rab27a effector coronin 3 controls endocytosis of secretory membrane in insulin-secreting cell lines

Rab27a is involved in the control of membrane traffic, a crucial step in the regulated secretion. Typically, the guanosine triphosphate (GTP)-bound form has been considered to be active and, therefore, searching for proteins binding to the GTP-form has been attempted to look for their effectors. Here, we have identified the actin-bundling protein coronin 3 as a novel Rab27a effector that paradoxically bound guanosine diphosphate (GDP)-Rab27a in the pancreatic β-cell line MIN6. Coronin 3 directly bound GDP-Rab27a through its β-propeller structure. The most important insulin secretagogue glucose promptly shifted Rab27a from the GTP- to GDP-bound form. Knockdown of coronin 3 by RNAi resulted in the inhibition of phogrin (an insulin-granule-associated protein) internalization and the uptake of FM4-64 (a marker of endocytosis). Similar results were reproduced by disruption of the coronin-3–GDP-Rab27a interaction with the dominant-negative coronin 3, and coexpression of the GDP-Rab27a mutant rescued these changes. Taken together, our results indicate that interaction of GDP-Rab27a and coronin 3 is important in stimulus-endocytosis coupling, and that GTP- and GDP-Rab27a regulates insulin membrane recycling at the distinct stages.

CRMP‐2 directly binds to cytoplasmic dynein and interferes with its activity

The active transport of proteins and organelles is critical for cellular organization and function in eukaryotic cells. A substantial portion of long‐distance transport depends on the opposite polarity of the kinesin and dynein family molecular motors to move cargo along microtubules. It is increasingly clear that many cargo molecules are moved bi‐directionally by both sets of motors; however, the regulatory mechanism that determines the directionality of transport remains unclear. We previously reported that collapsin response mediator protein‐2 (CRMP‐2) played key roles in axon elongation and neuronal polarization. CRMP‐2 was also found to associate with the anterograde motor protein Kinesin‐1 and was transported with other cargoes toward the axon terminal. In this study, we investigated the association of CRMP‐2 with a retrograde motor protein, cytoplasmic dynein. Immunoprecipitation assays showed that CRMP‐2 interacted with cytoplasmic dynein heavy chain. Dynein heavy chain directly bound to the N‐terminus of CRMP‐2, which is the distinct side of CRMP‐2’s kinesin light chain‐binding region. Furthermore, over‐expression of the dynein‐binding fragments of CRMP‐2 prevented dynein‐driven microtubule transport in COS‐7 cells. Given that CRMP‐2 is a key regulator of axon elongation, this interference with cytoplasmic dynein function by CRMP‐2 might have an important role in axon formation, and neuronal development.

Endogenous hydrogen sulfide protects pancreatic beta-cells from a high-fat diet-induced glucotoxicity and prevents the development of type 2 diabetes

Chronic exposure to high glucose induces the expression of cystathionine gamma-lyase (CSE), a hydrogen sulfide-producing enzyme, in pancreatic beta-cells, thereby suppressing apoptosis. The aim of this study was to examine the effects of hydrogen sulfide on the onset and development of type 2 diabetes. Middle-aged (6-month-old) wild-type (WT) and CSE knockout (CSE-KO) mice were fed a high-fat diet (HFD) for 8weeks. We determined the effects of CSE knockout on beta-cell function and mass in islets from these mice. We also analyzed changes in gene expression in the islets. After 8weeks of HFD, blood glucose levels were markedly increased in middle-aged CSE-KO mice, insulin responses were significantly reduced, and DNA fragmentation of the islet cells was increased. Moreover, expression of thioredoxin binding protein-2 (TBP-2, also known as Txnip) was increased. Administration of NaHS, a hydrogen sulfide donor, reduced TBP-2 gene levels in isolated islets from CSE-KO mice. Gene levels were elevated when islets were treated with the CSE inhibitor dl-propargylglycine (PPG). These results provide evidence that CSE-produced hydrogen sulfide protects beta-cells from glucotoxicity via regulation of TBP-2 expression levels and thus prevents the onset/development of type 2 diabetes.