Anikó Váradi

Cytoplasmic dynein regulates the subcellular distribution of mitochondria by controlling the recruitment of the fission factor dynamin-related protein-1.

While the subcellular organisation of mitochondria is likely to influence many aspects of cell physiology, its molecular control is poorly understood. Here, we have investigated the role of the retrograde motor protein complex, dynein-dynactin, in mitochondrial localisation and morphology. Disruption of dynein function, achieved in HeLa cells either by over-expressing the dynactin subunit, dynamitin (p50), or by microinjection of an anti-dynein intermediate chain antibody, resulted in (a) the redistribution of mitochondria to the nuclear periphery, and (b) the formation of long and highly branched mitochondrial structures. Suggesting that an alteration in the balance between mitochondrial fission and fusion may be involved in both of these changes, overexpression of p50 induced the translocation of the fission factor dynamin-related protein (Drp1) from mitochondrial membranes to the cytosol and microsomes. Moreover, a dominant-negative-acting form of Drp1 mimicked the effects of p50 on mitochondrial morphology, while wild-type Drp1 almost completely restored normal mitochondrial distribution in p50 over-expressing cells. Thus, the dynein/dynactin complex plays an unexpected role in the regulation of mitochondrial morphology in living cells, by controlling the recruitment of Drp1 to these organelles.

Involvement of conventional kinesin in glucose-stimulated secretory granule movements and exocytosis in clonal pancreatic beta-cells.

Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of insulin in response to glucose. At present, the molecular motors involved in this movement, and the mechanisms whereby they may be regulated, are undefined. To investigate the role of kinesin family members, we labelled densecore vesicles in clonal β-cells using an adenovirally expressed, vesicle-targeted green fluorescent protein (phogrin.EGFP), and employed immunoadsorption to obtain highly purified insulin-containing vesicles. Whereas several kinesin family members were expressed in this cell type, only conventional kinesin heavy chain (KHC) was detected in vesicle preparations. Expression of a dominant-negative KHC motor domain (KHCmut) blocked all vesicular movements with velocity> 0.4 μm second-1, which demonstrates that kinesin activity was essential for vesicle motility in live β-cells. Moreover, expression of KHCmut strongly inhibited the sustained, but not acute, stimulation of secretion by glucose. Finally, vesicle movement was stimulated by ATP dose-dependently in permeabilized cells, which suggests that glucose-induced increases in cytosolic [ATP] mediate the effects of the sugar in vivo, by enhancing kinesin activity. These data therefore provide evidence for a novel mechanism whereby glucose may enhance insulin release.

Developing oligodendrocytes express functional GABAB receptors that stimulate cell proliferation and migration

GABAB receptors (GABABRs) are involved in early events during neuronal development. The presence of GABABRs in developing oligodendrocytes has not been established. Using immunofluorescent co‐localization, we have identified GABABR proteins in O4 marker‐positive oligodendrocyte precursor cells (OPCs) in 4‐day‐old mouse brain periventricular white matter. In culture, OPCs, differentiated oligodendrocytes (DOs) and type 2 astrocytes (ASTs) express both the GABAB1abcdf and GABAB2 subunits of the GABABR. Using semiquantitative PCR analysis with GABABR isoform‐selective primers we found that the expression level of GABAB1abd was substantially higher in OPCs or ASTs than in DOs. In contrast, the GABAB2 isoform showed a similar level of expression in OPCs and DOs, and a significantly higher level in ASTs. This indicates that the expression of GABAB1 and GABAB2 subunits are under independent control during oligodendroglial development. Activation of GABABRs using the selective agonist baclofen demonstrated that these receptors are functionally active and negatively coupled to adenylyl cyclase. Manipulation of GABABR activity had no effect on OPC migration in a conventional agarose drop assay, whereas baclofen significantly increased OPC migration in a more sensitive transwell microchamber‐based assay. Exposure of cultured OPCs to baclofen increased their proliferation, providing evidence for a functional role of GABABRs in oligodendrocyte development. The presence of GABABRs in developing oligodendrocytes provides a new mechanism for neuronal–glial interactions during development and may offer a novel target for promoting remyelination following white matter injury.

Oligodendroglial metabotropic glutamate receptors are developmentally regulated and involved in the prevention of apoptosis

Oligodendrocytes (OLs) are responsible for axon myelination and are the principal cells targeted in preterm white matter injury. The cellular and molecular mechanisms involved in white matter development and immature OL injury are incompletely understood. Metabotropic glutamate receptors (mGluRs) modulate neuronal development and survival, and have recently been identified in oligodendrocyte progenitor cells (OPCs). Using the highly homogeneous CG‐4 OPC line and O4 marker‐immunoselected primary OLs, we established the differentiation stage‐specific expression profile of mGluR3 and mGluR5 mRNAs and proteins in the oligodendroglial lineage and type‐2‐astrocytes (ASTs). Our quantitative analysis indicated no changes in mGluR3, but a significant down‐regulation of mGluR5a mRNA and protein expression during differentiation of OPCs into OLs or ASTs. The down‐regulation of mGluR5a had functional consequences, with significantly fewer OLs and ASTs than OPCs responding to the group I mGluR agonist (RS)‐3,5‐dihydroxyphenylglycine with intracellular Ca2+ concentration oscillations. Neither stimulation nor inhibition of mGluR3 or mGluR5 altered OPC migration, suggesting that these receptors do not play prominent roles in the regulation of OPC motility. The activation of mGluR5 completely protected OPCs and substantially reduced staurosporine‐induced apoptosis in OLs. This suggests that the down‐regulation of mGluR5 in premyelinating OLs is likely to contribute to their increased vulnerability, and that the targeting of mGluR5 may be a potential therapeutic strategy for future development.

Translationally controlled tumour protein (TCTP) is a novel glucose-regulated protein that is important for survival of pancreatic beta cells.

Aims/hypothesisThis study used proteomics and biochemical approaches to identify novel glucose-regulated proteins and to unveil their role in pancreatic beta cell function. Translationally controlled tumour protein (TCTP) was identified to be one such protein, and further investigations into its function and regulation were carried out.MethodsGlobal protein profiling of beta cell homogenates following glucose stimulation was performed using two-dimensional gel electrophoresis. Proteins were identified by mass spectroscopy analysis. Immunoblotting was used to investigate alterations in TCTP protein levels in response to glucose stimulation or cell stress induced by palmitate. To investigate the biological function of TCTP, immunolocalisation, gene knockdown and overexpression of Tctp (also known as Tpt1) were performed. Apoptosis was measured in Tctp knockdown or Tctp-overexpressing cells. Glucose-stimulated insulin secretion was carried out in Tctp knockdown cells.ResultsTCTP was identified as a novel glucose-regulated protein, the level of which is increased at stimulatory glucose concentration. Glucose also induced TCTP dephosphorylation and its partial translocation to the mitochondria and the nucleus. TCTP protein levels were downregulated in response to cell stress induced by palmitate or thapsigargin treatments. Gene knockdown by small interfering RNA led to increased apoptosis, whereas overproduction of TCTP prevented palmitate-induced cell death.Conclusions/interpretationRegulation of TCTP protein levels by glucose is likely to be an important cyto-protective mechanism for pancreatic beta cells against damage caused by hyperglycaemia. In contrast, high concentration of palmitate causes cell stress, reduction in TCTP levels and consequently reduced cell viability. Our results imply that TCTP levels influence the sensitivity of beta cells to apoptosis.

Molecular Mechanism of Myosin Va Recruitment to Dense Core Secretory Granules

The brain‐spliced isoform of Myosin Va (BR‐MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide‐producing cells. The molecular composition of the protein complex that recruits BR‐MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG‐associated proteins granuphilin‐a/b (Gran‐a/b), BR‐MyoVa and Rab27a, a member of the Rab family of GTPases. Gran‐a/b–BR‐MyoVa interaction is direct, involves regions downstream of the Rab27‐binding domain, and the C‐terminal part of Gran‐a determines exon specificity. MyoVa and Gran‐a/b are partially colocalised on SGs and disruption of Gran‐a/b–BR‐MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient‐stimulated hormone secretion in pancreatic beta‐cells. These results indicate the existence of at least another binding partner of BR‐MyoVa that was identified as rabphilin‐3A (Rph‐3A). BR‐MyoVa–Rph‐3A interaction is also direct and enhanced when secretion is activated. The BR‐MyoVa–Rph‐3A and BR‐MyoVa–Gran‐a/b complexes are linked to a different subset of SGs, and simultaneous inhibition of these complexes nearly completely blocks stimulated hormone release. This study demonstrates that multiple binding partners of BR‐MyoVa regulate SG transport, and this molecular mechanism is universally used by neuronal, endocrine and neuroendocrine cells.

MyRIP interaction with MyoVa on secretory granules is controlled by the cAMP-PKA pathway

Myosin- and Rab-interacting protein is not a classic receptor for MyoVa on large, dense-core secretory granules (SGs), but it aids in PKA-dependent phosphorylation of MyoVa-associated proteins on SGs in endocrine and neuroendocrine cells.

mGluR5 is involved in dendrite differentiation and excitatory synaptic transmission in NTERA2 human embryonic carcinoma cell-derived neurons

The pluripotent human embryonic carcinoma cell line NTERA2 readily differentiates into neurons when exposed to retinoic acid in vitro. These neurons show characteristic morphology with long processes and they express neuronal markers TUJ-1 and NeuN. NTERA2-derived neurons can regulate Ca2+ signalling through ionotropic glutamate (iGluR) and muscarinic receptors (mAChRs). Little is known, however, about the role of metabotropic glutamate receptors (mGluRs) in these neurons. Here we show that NTERA2-derived neurons express functional mGluR5, which is involved in Ca2+ signalling. Blocking mGluR5 activity at early stages of differentiation leads to fewer dendrites and a reduction in miniature excitatory postsynaptic currents (mEPSCs). Furthermore, cells cultured in the presence of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) show reduced N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ mobilisation but increased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor Ca2+ permeability. During normal neuronal development, the edited GluR2 renders AMPARs Ca2+ impermeable. The increased Ca2+ permeability of AMPARs in MPEP-treated neurons is due to the reduced expression of GluR2 subunit protein. Thus, mGluR5 activity at early stages of differentiation is likely to play a role in the development of multipotent cell-derived neurons.

Hb Fontainebleau (HBA2: c.64G > C) in the United Arab Emirates

Abstract Hb Fontainebleau (HBA2: c.64G > C) is a rare α-globin variant, which has previously been described in only 10 individuals worldwide. We report here 12 additional cases identified in our laboratory. These included the first case of a homozygosity for Hb Fontainebleau and cases in which Hb Fontainebleau occurred in combination with deletional and nondeletional α-thalassemia (α-thal). The prevalence of Hb Fontainebleau in the samples submitted to our laboratory for premarital hemoglobinopathy screening was 0.24%, the highest reported prevalence to date, indicating that this is a comparatively common variant in the United Arab Emirates (UAE).

Detection of three closely located single nucleotide polymorphisms in the EAAT2 promoter: comparison of single-strand conformational polymorphism (SSCP), pyrosequencing and Sanger sequencing

BackgroundSingle-strand conformational polymorphism (SSCP) is still a frequently used genotyping method across different fields for the detection of single nucleotide polymorphisms (SNPs) due to its simplicity, requirement for basic equipment accessible in most laboratories and low cost. This technique was previously used to detect rs4354668:A > C (g.-181A > C) SNP in the promoter of astroglial glutamate transporter (EAAT2) and the same approach was initially used here to investigate this promoter region in a cohort of newborns.ResultsUnexpectedly, four distinct DNA migration patterns were identified by SSCP. Sanger sequencing revealed two additional SNPs: g.-200C > A and g.-168C > T giving a rise to a total of ten EAAT2 promoter variants. SSCP failed to distinguish these variants reliably and thus pyrosequencing assays were developed. g.-168C > T was found in heterozygous form in one infant only with minor allele frequency (MAF) of 0.0023. In contrast, g.-200C > A and -181A > C were more common (with MAF of 0.46 and 0.49, respectively) and showed string evidence of linkage disequilibrium (LD). In a systematic comparison, 16% of samples were miss-classified by SSCP with 25-31% errors in the identification of the wild-type and homozygote mutant genotypes compared to pyrosequencing or Sanger sequencing. In contrast, SSCP and pyrosequencing of an unrelated single SNP (rs1835740:C > T), showed 94% concordance.ConclusionOur data suggest that SSCP cannot always detect reliably several closely located SNPs. Furthermore, caution is needed in the interpretation of the association studies linking only one of the co-inherited SNPs in the EAAT2 promoter to human diseases.