ORP9-PH domain-based fluorescent reporters for visualizing phosphatidylinositol 4-phosphate dynamics in living cells.

Fluorescent reporters that visualize phosphatidylinositol 4-phosphate (PI4P) in living cells are indispensable to elucidate the roles of this fundamental lipid in cell physiology. However, currently available PI4P reporters have limitations, such as Golgi-biased localization and low detection sensitivity. Here, we present a series of fluorescent PI4P reporters based on the pleckstrin homology (PH) domain of oxysterol-binding protein-related protein 9 (ORP9). We show that the green fluorescent protein AcGFP1-tagged ORP9-PH domain can be used as a fluorescent PI4P reporter to detect cellular PI4P across its wide distribution at multiple cellular locations, including the plasma membrane (PM), Golgi, endosomes, and lysosomes with high specificity and contrast. We also developed blue, red, and near-infrared fluorescent PI4P reporters suitable for multicolor fluorescence imaging experiments. Finally, we demonstrate the utility of the ORP9-PH domain-based reporter to visualize dynamic changes in the PI4P distribution and level in living cells upon synthetic ER-PM membrane contact manipulation and GPCR stimulation. This work offers a new set of genetically encoded fluorescent PI4P reporters that are practically useful for the study of PI4P biology.

Reduced chondroitin sulfate content prevents diabetic neuropathy through transforming growth factor-ß signaling suppression.

Diabetic neuropathy (DN) is a major complication of diabetes mellitus. Chondroitin sulfate (CS) is one of the most important extracellular matrix components and is known to interact with various diffusible factors; however, its role in DN pathology has not been examined. Therefore, we generated CSGalNAc-T1 knockout (T1KO) mice, in which CS levels were reduced. We demonstrated that diabetic T1KO mice were much more resistant to DN than diabetic wild-type (WT) mice. We also found that interactions between pericytes and vascular endothelial cells were more stable in T1KO mice. Among the RNA-seq results, we focused on the transforming growth factor ß signaling pathway and found that the phosphorylation of Smad2/3 was less upregulated in T1KO mice than in WT mice under hyperglycemic conditions. Taken together, a reduction in CS level attenuates DN progression, indicating that CS is an important factor in DN pathogenesis.

Phosphatidylserine turns the gears of phospholipids in B cell lymphoma.

Phosphatidylserine levels and distribution are tightly controlled by dedicated enzymes at the ER and plasma membrane. Nakatsu and Kawasaki discuss new work by Aoki and colleagues (https://doi.org/10.1083/jcb.202212074), which reveals an acute reliance on phosphatidylserine synthesis in B cell lymphomas needed to prevent aberrant B cell receptor activation and ensuing apoptosis.

Very-long-chain fatty acids are crucial to neuronal polarity by providing sphingolipids to lipid rafts.

Fatty acids have long been considered essential to brain development; however, the involvement of their synthesis in nervous system formation is unclear. We generate mice with knockout of GPSN2, an enzyme for synthesis of very-long-chain fatty acids (VLCFAs) and investigate the effects. Both GPSN2-/- and GPSN2+/- mice show abnormal neuronal networks as a result of impaired neuronal polarity determination. Lipidomics of GPSN2-/- embryos reveal that ceramide synthesis is specifically inhibited depending on FA length; namely, VLCFA-containing ceramide is reduced. We demonstrate that lipid rafts are highly enriched in growth cones and that GPSN2+/- neurons lose gangliosides in their membranes. Application of C24:0 ceramide, but not C16:0 ceramide or C24:0 phosphatidylcholine, to GPSN2+/- neurons rescues both neuronal polarity determination and lipid-raft density in the growth cone. Taken together, our results indicate that VLCFA synthesis contributes to physiological neuronal development in brain network formation, in particular neuronal polarity determination through the formation of lipid rafts.

PI4P/PS countertransport by ORP10 at ER-endosome membrane contact sites regulates endosome fission.

Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER-endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER-endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER-endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.

Functions of Oxysterol-Binding Proteins at Membrane Contact Sites and Their Control by Phosphoinositide Metabolism.

Lipids must be correctly transported within the cell to the right place at the right time in order to be fully functional. Non-vesicular lipid transport is mediated by so-called lipid transfer proteins (LTPs), which contain a hydrophobic cavity that sequesters lipid molecules. Oxysterol-binding protein (OSBP)-related proteins (ORPs) are a family of LTPs known to harbor lipid ligands, such as cholesterol and phospholipids. ORPs act as a sensor or transporter of those lipid ligands at membrane contact sites (MCSs) where two different cellular membranes are closely apposed. In particular, a characteristic functional property of ORPs is their role as a lipid exchanger. ORPs mediate counter-directional transport of two different lipid ligands at MCSs. Several, but not all, ORPs transport their lipid ligand from the endoplasmic reticulum (ER) in exchange for phosphatidylinositol 4-phosphate (PI4P), the other ligand, on apposed membranes. This ORP-mediated lipid "countertransport" is driven by the concentration gradient of PI4P between membranes, which is generated by its kinases and phosphatases. In this review, we will discuss how ORP function is tightly coupled to metabolism of phosphoinositides such as PI4P. Recent progress on the role of ORP-mediated lipid transport/countertransport at multiple MCSs in cellular functions will be also discussed.

Distinct effects of chondroitin sulfate on hematopoietic cells and the stromal microenvironment in bone marrow hematopoiesis.

The bone marrow (BM) microenvironment, known as the BM niche, regulates hematopoiesis but is also affected by interactions with hematopoietic cells. Recent evidence indicates that extracellular matrix components are involved in these interactions. Chondroitin sulfate (CS), a glycosaminoglycan, is a major component of the extracellular matrix; however, it is not known whether CS has a physiological role in hematopoiesis. Here, we analyzed the functions of CS in hematopoietic and niche cells. CSGalNAcT1, which encodes CS N-acetylgalactosaminyltransferase-1 (T1), a key enzyme in CS biosynthesis, was highly expressed in hematopoietic stem and progenitor cells (HSPCs) and endothelial cells (ECs), but not in mesenchymal stromal cells (MSCs) in BM. In T1 knockout (T1KO) mice, a greater number of HSPCs existed compared with the wild-type (WT), but HSPCs from T1KO mice showed significantly impaired repopulation in WT recipient mice on serial transplantation. RNA sequence analysis revealed the activation of IFN-α/ß signaling and endoplasmic reticulum stress in T1KO HSPCs. In contrast, the number of WT HSPCs repopulated in T1KO recipient mice was larger than that in WT recipient mice after serial transplantation, indicating that the T1KO niche supports repopulation of HSPCs better than the WT niche. There was no obvious difference in the distribution of vasculature and MSCs between WT and T1KO BM, suggesting that CS loss alters vascular niche functions without affecting its structure. Our results revealed distinct roles of CS in hematopoietic cells and BM niche, indicating that crosstalk between these components is important to maintain homeostasis in BM.

Neuronal Signaling Involved in Neuronal Polarization and Growth: Lipid Rafts and Phosphorylation.

Neuronal polarization and growth are developmental processes that occur during neuronal cell differentiation. The molecular signaling mechanisms involved in these events in in vivo mammalian brain remain unclear. Also, cellular events of the neuronal polarization process within a given neuron are thought to be constituted of many independent intracellular signal transduction pathways (the "tug-of-war" model). However, in vivo results suggest that such pathways should be cooperative with one another among a given group of neurons in a region of the brain. Lipid rafts, specific membrane domains with low fluidity, are candidates for the hotspots of such intracellular signaling. Among the signals reported to be involved in polarization, a number are thought to be present or translocated to the lipid rafts in response to extracellular signals. As part of our analysis, we discuss how such novel molecular mechanisms are combined for effective regulation of neuronal polarization and growth, focusing on the significance of the lipid rafts, including results based on recently introduced methods.

Phosphoproteomic and bioinformatic methods for analyzing signaling in vertebrate axon growth and regeneration.

Phosphorylation is the most important post-translational modification of proteins in many cells, including neurons. Phosphoproteomics is a relatively new technique for comprehensively identifying phosphorylation sites in the whole proteome of a given system. We applied this method to developmental neurobiology research to understand the signaling pathways that regulate the mammalian growth cone, which is formed at the tips of developing neurites to ensure accurate neuronal network formation. Using this powerful technique, we identified at least four phosphorylation sites tightly associated with axon growth. Because phosphoproteomic results include relatively large numbers of phosphopeptides, the data are typically analyzed using bioinformatics. We utilized three bioinformatics tools to identify the responsible protein kinases, the putative functions of the phosphorylated protein groups, and the evolutional aspects of the phosphorylated proteins. Collectively, these data indicate phosphoproteomics is a cutting-edge tool for neuroscience research.

Growth Cone Phosphoproteomics Reveals that GAP-43 Phosphorylated by JNK Is a Marker of Axon Growth and Regeneration.

Neuronal growth cones are essential for nerve growth and regeneration, as well as for the formation and rearrangement of the neural network. To elucidate phosphorylation-dependent signaling pathways and establish useful molecular markers for axon growth and regeneration, we performed a phosphoproteomics study of mammalian growth cones, which identified >30,000 phosphopeptides of ∼1,200 proteins. The phosphorylation sites were highly proline directed and primarily MAPK dependent, owing to the activation of JNK, suggesting that proteins that undergo proline-directed phosphorylation mediate nerve growth in the mammalian brain. Bioinformatics analysis revealed that phosphoproteins were enriched in microtubules and the cortical cytoskeleton. The most frequently phosphorylated site was S96 of GAP-43 (growth-associated protein 43-kDa), a vertebrate-specific protein involved in axon growth. This previously uncharacterized phosphorylation site was JNK dependent. S96 phosphorylation was specifically detected in growing and regenerating axons as the most frequent target of JNK signaling; thus it represents a promising new molecular marker for mammalian axonal growth and regeneration.

Abnormalities in perineuronal nets and behavior in mice lacking CSGalNAcT1, a key enzyme in chondroitin sulfate synthesis.

Chondroitin sulfate (CS) is an important glycosaminoglycan and is mainly found in the extracellular matrix as CS proteoglycans. In the brain, CS proteoglycans are highly concentrated in perineuronal nets (PNNs), which surround synapses and modulate their functions. To investigate the importance of CS, we produced and precisely examined mice that were deficient in the CS synthesizing enzyme, CSGalNAcT1 (T1KO). Biochemical analysis of T1KO revealed that loss of this enzyme reduced the amount of CS by approximately 50% in various brain regions. The amount of CS in PNNs was also diminished in T1KO compared to wild-type mice, although the amount of a major CS proteoglycan core protein, aggrecan, was not changed. In T1KO, we observed abnormalities in several behavioral tests, including the open-field test, acoustic startle response, and social preference. These results suggest that T1 is important for plasticity, probably due to regulation of CS-dependent PNNs, and that T1KO is a good model for investigation of PNNs.

Elasticity of the tibial nerve assessed by sonoelastography was reduced before the development of neuropathy and further deterioration associated with the severity of neuropathy in patients with type 2 diabetes.

AIMS/INTRODUCTION: To measure the elasticity of the tibial nerve using sonoelastography, and to associate it with diabetic neuropathy severity, the cross-sectional area of the tibial nerve and neurophysiological findings in type 2 diabetic patients. MATERIALS AND METHODS: The elasticity of the tibial nerve was measured as the tibial nerve:acoustic coupler strain ratio using high-resolution ultrasonography in 198 type 2 diabetic patients stratified into subgroups by neuropathy severity, and 29 control participants whose age and sex did not differ from the diabetic subgroups. RESULTS: The elasticity of the tibial nerve in patients without neuropathy (P < 0.001) was reduced compared with controls (0.76 ± 0.023), further decreasing (0.655 ± 0.014 to 0.414 ± 0.018) after developing neuropathy. The cut-off value of elasticity of the tibial nerve that suggested the presence of neuropathy was 0.558. The area under the curve (0.829) was greater than that for the cross-sectional area (0.612). The cross-sectional area of the tibial nerve in diabetic patients without neuropathy (6.11 ± 0.13 mm(2)) was larger than that in controls (4.84 ± 0.16 mm(2)), and increased relative to neuropathy severity (P < 0.0001). The elasticity of the tibial nerve was negatively associated with neuropathy severity (P < 0.0001), cross-sectional area (P = 0.002) and 2000 Hz current perception threshold (P = 0.011), and positively associated with nerve conduction velocities (P < 0.0001). CONCLUSIONS: Determining the elasticity of the tibial nerve in type 2 diabetic patients could reveal early biomechanical changes that were likely caused by thickened fibrous sheaths of peripheral nerves, and might be a novel tool for characterizing diabetic neuropathy.

Morphological changes of the peripheral nerves evaluated by high-resolution ultrasonography are associated with the severity of diabetic neuropathy, but not corneal nerve fiber pathology in patients with type 2 diabetes.

AIMS/INTRODUCTION: To evaluate the morphological changes of the median and posterior tibial nerve using high-resolution ultrasonography, and the corneal C fiber pathology by corneal confocal microscopy in type 2 diabetic patients. MATERIALS AND METHODS: The cross-sectional area, hypoechoic area and maximum thickness of the nerve fascicle of both nerves were measured by high-resolution ultrasonography in 200 type 2 diabetic patients, stratified by the severity of diabetic neuropathy, and in 40 age- and sex-matched controls. These parameters were associated with corneal C fiber pathology visualized by corneal confocal microscopy, neurophysiological tests and severity of diabetic neuropathy. RESULTS: The cross-sectional area, hypoechoic area and maximum thickness of the nerve fascicle of both nerves in patients without diabetic neuropathy were larger than those in control subjects (P < 0.05 to P < 0.001), and further increased relative to the severity of neuropathy (P < 0.0001). All morphological changes of both nerves were negatively associated with motor and sensory nerve conduction velocity (P = 0.01 to P < 0.0001), and directly associated with 2,000-Hz current perception threshold (P = 0.009 to P < 0.001). The significant corneal C fiber pathology occurred before developing the neuropathy, and deteriorated only in patients with the most severe neuropathy. The association between the morphological changes of both nerves and corneal C fiber pathology was poor. CONCLUSIONS: The morphological changes in peripheral nerves of type 2 diabetic patients were found before the onset of neuropathy, and were closely correlated with the severity of diabetic neuropathy, but not with corneal C fiber pathology.

Correlation between sudomotor function, sweat gland duct size and corneal nerve fiber pathology in patients with type 2 diabetes mellitus.

AIMS/INTRODUCTION: To study the correlation between sudomotor function, sweat gland duct size and corneal nerve fiber pathology in type 2 diabetes. MATERIALS AND METHODS: Sudomotor function was quantified by Neuropad test, and sweat gland duct and corneal nerve fibers were visualized by confocal microscopy in 78 patients with type 2 diabetes stratified by diabetic neuropathy and 28 control participants. RESULTS: In patients with diabetic neuropathy, sudomotor function, as judged by the time required for complete color change of a Neuropad, was impaired compared with that of controls (P < 0.0001), thereby showing deterioration was related to the severity of diabetic neuropathy (P < 0.0001). Sweat gland ducts were smaller in patients without neuropathy than in controls (P < 0.0001), and further shrinking was seen in patients with severe diabetic neuropathy (P < 0.05). Patients without diabetic neuropathy showed reduced density and length (P < 0.001) of corneal nerve fibers and beading frequency (P < 0.0001), and increased tortuosity (P < 0.0001) compared with controls, and these changes deteriorated in patients with severe diabetic neuropathy. Sudomotor function was negatively associated with corneal nerve fibers (P < 0.002) and branches (P < 0.01), and influenced by the severity of diabetic neuropathy (P < 0.0001); sweat gland duct size correlated with serum triglycerides (P < 0.02), uric acid (P < 0.01), corneal nerve branch (P < 0.03), sudomotor function (P < 0.03) and severity of neuropathy (P < 0.03). CONCLUSIONS: Type 2 diabetic patients had sudomotor dysfunction and smaller sweat gland ducts compared with controls. The stage of diabetic neuropathy and corneal nerve fiber pathology were independent predictors of sudomotor dysfunction, and serum triglycerides, uric acid, corneal nerve branch, stage of diabetic neuropathy and sudomotor function were predictors of sweat gland duct size.

Involvement of IQGAP family proteins in the regulation of mammalian cell cytokinesis.

IQGAP family proteins, comprising IQGAP1, -2, and -3 in mammals, are involved in diverse ranges of cellular processes such as adhesion and migration. IQGAP proteins in yeast also play important roles in cytokinesis. However, the involvement of IQGAP proteins in cytokinesis in mammals remains unaddressed. In this study, we showed that IQGAP3 specifically localized to the equatorial cortex at anaphase, whereas IQGAP1 localized to the cell cortex uniformly and IQGAP2 was unexpressed in HeLa cells. IQGAP3, but neither IQGAP1 nor -2, was able to interact with anillin, which was required for the localization of IQGAP3 to the contractile ring. The suppressed expression of IQGAP3 inhibited the completion of cleavage furrow ingression and led to the multinucleation of cells. The suppression of IQGAP1 also had similar inhibitory effects on cytokinesis, and the simultaneous suppression of IQGAP1 and -3 induced more severe effects. The localization of anillin and RhoA to the contractile ring was impaired by the suppression of IQGAP1 and -3, whereas their upstream regulators, the centralspindlin complex and Ect2, remained unaffected. These results suggested that mammalian IQGAP proteins may play a role in cytokinesis by regulating the localization of key cytokinesis regulatory proteins to the contractile apparatus during mitosis.

Morphometric features of corneal epithelial basal cells, and their relationship with corneal nerve pathology and clinical factors in patients with type 2 diabetes.

AIMS/INTRODUCTION: We compared the morphometric features of corneal epithelial basal cells between patients with type 2 diabetes mellitus and healthy controls, and analyzed the relationship of these features with corneal nerve fiber pathology and clinical factors in the patients. MATERIALS AND METHODS: Corneal epithelial basal cells and corneal nerve fibers were visualized by corneal confocal microscopy in 75 patients with type 2 diabetes and 42 age-matched controls. Density, area and area variability of corneal epithelial basal cells, as well as the width of the intercellular space between neighboring cells, were evaluated for both groups. RESULTS: Patients showed decreased density (P < 0.02) and area (P < 0.0001), larger area variability (P < 0.0001) and a wider intercellular space (P < 0.0001) compared with controls. Density correlated inversely with area (P < 0.0001), width of intercellular space (P < 0.03) and beading frequency (P < 0.03), whereas it correlated directly with prothrombin time (P < 0.002) and activated partial thromboplastin time (P < 0.03). Area correlated inversely with duration of diabetes (P < 0.05) and coefficient of variation of area (P < 0.01), whereas it correlated directly with beading frequency (P < 0.05). Area variability correlated inversely with area (P < 0.01) and prothrombin time (P < 0.01), whereas it correlated directly with fibrinogen level (P < 0.0001). CONCLUSIONS: Type 2 diabetes induces morphometric changes in corneal epithelial basal cells; this seems to be related to the morbid period of diabetes, beading frequency of corneal nerve fibers and blood coagulation state.

Cell-autonomous enhancement of glutamate-uptake by female astrocytes.

Since gonadal female hormones act on and protect neurons, it is well known that the female brain is less vulnerable to stroke or other brain insults than the male brain. Although glial functions have been shown to affect the vulnerability of the brain, little is known if such a sex difference exists in glia, much less the mechanism that might cause gender-dependent differences in glial functions. In this study, we show that in vitro astrocytes obtained from either female or male pups show a gonadal hormone-independent phenotype that could explain the gender-dependent vulnerability of the brain. Female spinal astrocytes cleared more glutamate by GLAST than male ones. In addition, motoneurons seeded on female spinal astrocytes were less vulnerable to glutamate than those seeded on male ones. It is suggested that female astrocytes uptake more glutamate and reveal a stronger neuroprotective effect against glutamate than male ones. It should be noted that such an effect was independent of gonadal female hormones, suggesting that astrocytes have cell-autonomous regulatory mechanisms by which they transform themselves into appropriate phenotypes.

Corneal nerve fiber pathology in Japanese type 1 diabetic patients and its correlation with antecedent glycemic control and blood pressure.

UNLABELLED: Aims/Introduction: Morphological changes to corneal C-fibers in Japanese type 1 diabetic patients were visualized by corneal confocal microscopy (CCM). The effects of prior glycemic control and blood pressure on morphological parameters were clarified. MATERIALS AND METHODS: Corneal nerve fibers were visualized by CCM in 38 Japanese type 1 diabetic patients (14 with and 24 without neuropathy) and 38 controls. Morphological parameters were compared and related to annual mean HbA1c, blood pressure, and serum lipid levels of previous years prior to CCM examination. RESULTS: Compared with controls, diabetic patients had reduced corneal nerve fiber length (CNFL; 9.80 ± 0.38 vs 13.65 ± 0.88 mm/mm(2); P < 0.001), reduced density (CNFD; 25.32 ± 1.04 vs 36.62 ± 2.37/mm(2); P < 0.0005), lower frequency of beading (22.38 ± 0.73 vs 30.44 ± 1.03/0.1 mm; P < 0.0001), and increased tortuosity (3.13 ± 0.09 vs 1.74 ± 0.06; P < 0.0001). These changes were found in patients without neuropathy. There was no difference in nerve branches between controls and diabetic patients. The mean annual HbA1c level for the 7-10 years prior to CCM examination was an independent predictor of reduced CNFL and CNFD; HbA1c levels obtained 1-3 months and 1 year prior to CCM, as well as blood pressure 3, 5, and 6 years prior to CCM, were independent predictors of reduced beading frequency. CONCLUSIONS: Corneal confocal microscopy is a novel, noninvasive technique to evaluate morphological changes of corneal C-fibers in type 1 diabetes. Antecedent hyperglycemia and blood pressure have different time-dependent effects on CNFL and CNFD and the frequency of beading. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00157.x, 2011).