Fourteen novel human members of mitochondrial solute carrier family 25 (SLC25) widely expressed in the central nervous system

Members of the solute carrier family 25 (SLC25) are known to transport molecules over the mitochondrial membrane. In this paper we present 14 novel members of SLC25 family in human. These were provided with following gene symbols by the HGNC: SLC25A32, SLC25A33, SLC25A34, SLC25A35, SLC25A37, SLC25A38, SLC25A39, SLC25A40, SLC25A41, SLC25A42, SLC25A43, SLC25A44, SLC25A45, and SLC25A46. We also identified the orthologues for these genes in rat and mouse. Moreover, we found yeast orthologues for 9 of these genes and show that the predicted substrate binding residues are highly conserved in the human and yeast proteins. We performed a comprehensive tissue localization study for 9 of these genes on a panel of 30 rat tissues with quantitative real-time polymerse chain reaction. We detected their mRNA in a wide number of tissues, both in brain and in periphery. This study provides an overall roadmap of the repertoire of the SLC25 family in mammals, showing that there are at least 46 genes in the human genome coding for mitochondrial transporters.     

Pombe Cdc15 homology proteins: regulators of membrane dynamics and the actin cytoskeleton

Pombe Cdc15 homology (PCH) proteins have emerged in many species as important coordinators of signalling pathways that regulate actomyosin assembly and membrane dynamics. For example, the prototype PCH protein, Cdc15p of Schizosaccharomyces pombe, has a role in assembly of the contractile ring, which is needed to separate dividing cells. Recently, mammalian PCH proteins have been found to bind phospholipids and to participate in membrane deformation. These findings suggest that PCH proteins are crucial linkers of membrane dynamics and actin polymerization, for example, during the internalization of transmembrane receptors. Intriguingly, some members of the PCH protein family are mutated in neurodegenerative and inflammatory diseases, which has implications for the identification of cures for such disorders.     

Members of the CIP4 family of proteins participate in the regulation of platelet‐derived growth factor receptor‐β‐dependent actin reorganization and migration

Background information. The F‐BAR {Fes/CIP4 [Cdc42 (cell division cycle 42)‐interacting protein 4] homology and BAR (Bin/amphiphysin/Rvs)} proteins have emerged as important co‐ordinators of signalling pathways that regulate actin assembly and membrane dynamics. The presence of the F‐BAR domain is the hallmark of this family of proteins and the CIP4 (Cdc42‐interacting protein 4) was one of the first identified vertebrate F‐BAR proteins. There are three human CIP4 paralogues, namely CIP4, FBP17 (formin‐binding protein 17) and Toca‐1 (transducer of Cdc42‐dependent actin assembly 1). The CIP4‐like proteins have been implicated in Cdc42‐dependent actin reorganization and in regulation of membrane deformation events visible as tubulation of lipid bilayers. Results. We performed side‐by‐side analyses of the three CIP4 paralogues. We found that the three CIP4‐like proteins vary in their effectiveness to catalyse membrane tubulation and actin reorganization. Moreover, we show that the CIP4‐dependent membrane tubulation is enhanced in the presence of activated Cdc42. Some F‐BAR members have been shown to have a role in the endocytosis of the EGF (epidermal growth factor) receptor and this prompted us to study the involvement of the CIP4‐like proteins in signalling of the PDGFRβ [PDGF (platelet‐derived growth factor) β‐receptor]. We found that knock‐down of CIP4‐like proteins resulted in a prolonged formation of PDGF‐induced dorsal ruffles, as well as an increased PDGF‐dependent cell migration. This was most likely a consequence of a sustained PDGFRβ activation caused by delayed internalization of the receptor in the cells treated with siRNA (small interfering RNA) specific for the CIP4‐like proteins. Conclusions. Our findings show that CIP4‐like proteins induced membrane tubulation downstream of Cdc42 and that they have important roles in PDGF‐dependent actin reorganization and cell migration by regulating internalization and activity of the PDGFRβ. Moreover, the results suggest an important role for the CIP4‐like proteins in the regulation of the activity of the PDGFRβ.     

Cholesterol loss enhances TrkB signaling in hippocampal neurons aging in vitro

Binding of the neurotrophin brain-derived neurotrophic factor (BDNF) to the TrkB receptor is a major survival mechanism during embryonic development. In the aged brain, however, BDNF levels are low, suggesting that if TrkB is to play a role in survival at this stage additional mechanisms must have developed. We here show that TrkB activity is most robust in the hippocampus of 21-d-old BDNF-knockout mice as well as in old, wild-type, and BDNF heterozygous animals. Moreover, robust TrkB activity is evident in old but not young hippocampal neurons differentiating in vitro in the absence of any exogenous neurotrophin and also in neurons from BDNF −/− embryos. Age-associated increase in TrkB activity correlated with a mild yet progressive loss of cholesterol. This, in turn, correlated with increased expression of the cholesterol catabolic enzyme cholesterol 24-hydroxylase. Direct cause–effect, cholesterol loss–high TrkB activity was demonstrated by pharmacological means and by manipulating the levels of cholesterol 24-hydroxylase. Because reduced levels of cholesterol and increased expression of choleseterol-24-hydroxylase were also observed in the hippocampus of aged mice, changes in cellular cholesterol content may be used to modulate receptor activity strength in vivo, autonomously or as a way to complement the natural decay of neurotrophin production.     

Regulation of lipid composition in Acholeplasma laidlawii and Escherichia coli membranes: NMR studies of lipid lateral diffusion at different growth temperatures

Lipid lateral diffusion coefficients have been directly determined by pulsed field gradient NMR spectroscopy on macroscopically aligned, fully hydrated lamellar phases containing dimyristoylphosphatidylcholine and total lipid extracts from Acholeplasma laidlawii and Escherichia coli. The temperature dependence of the diffusion coefficient was of the Arrhenius type in the temperature interval studied. The sharp increase in the diffusion coefficient at the growth temperature of E. coli obtained by FRAP measurements, using a fluorescent probe molecule (Jin, A. J., Edidin, M., Nossal, R., and Gershfeld, N. L. (1999) Biochemistry 38, 13275-13278), was not observed. Thus, we conclude that the lipid structural properties (i.e., those affecting the lipid phase behavior), rather than the lipid dynamics, are involved in the adjustment of the membrane lipid composition. Further support for this conclusion is given by the finding that lipid extracts from A. laidlawii grown at different temperatures have about the same diffusion coefficients. Finally, the lipid lateral diffusion in bilayers of phospholipids was found to be much faster than that in bilayers of mainly glucolipids, which can be understood in terms of a free volume theory for the diffusion process.     

Neurturin is a neuritogenic but not a survival factor for developing and adult central noradrenergic neurons

Noradrenergic neurons of the locus coeruleus (LC) express the receptor tyrosine kinase c-ret, which binds ligands of the glial cell line-derived neurotrophic factor (GDNF) family. In the present study, we evaluated the function of neurturin (NTN), a GDNF family ligand whose function on LC neurons is unknown. Interestingly, we found that tyrosine hydroxylase (TH)-positive neurons in the LC express both GFRalpha1 and 2 receptors in a developmentally regulated fashion, suggesting a function for their preferred ligands: GDNF and NTN, respectively. Moreover, our results show that NTN mRNA expression is developmentally down-regulated in the LC and peaks in the postnatal hippocampus and cerebral cortex, during the target innervation period. In order to examine the function of NTN, we next performed LC primary cultures, and found that neither GDNF nor NTN promoted the survival of TH-positive neurons. However, both factors efficiently induced neurite outgrowth in noradrenergic neurons (147% and 149% over controls, respectively). Similarly, grafting of fibroblast cell lines engineered to express high levels of NTN did not prevent the loss of LC noradrenergic neurons in a 6-hydroxydopamine (6-OHDA) lesion model, but induced the sprouting of TH-positive cells. Thus our findings show that NTN does not promote the survival of LC noradrenergic neurons, but induces neurite outgrowth in developing noradrenergic neurons in vitro and in a model of neurodegeneration in vivo. These data, combined with data in the literature, suggest that GDNF family ligands are able to independently regulate neuronal survival and/or neuritogenesis.     

Atypical Rho GTPases have roles in mitochondrial homeostasis and apoptosis

The human genomic sequencing effort has revealed the presence of a large number of Rho GTPases encoded by the human genome. Here we report the characterization of a new family of Rho GTPases with atypical features. These proteins, which were called Miro-1 and Miro-2 (for mitochondrial Rho), have tandem GTP-binding domains separated by a linker region containing putative calcium-binding EF hand motifs. Genes encoding Miro-like proteins were found in several eukaryotic organisms from Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster to mammals, indicating that these genes evolved early during evolution. Immunolocalization experiments, in which transfected NIH3T3 and COS 7 cells were stained for ectopically expressed Miro as well as for the endogenous Miro-1 protein, showed that Miro was present in mitochondria. Interestingly, overexpression of a constitutively active mutant of Miro-1 (Miro-1/Val-13) induced an aggregation of the mitochondrial network and resulted in an increased apoptotic rate of the cells expressing activated Miro-1. These data indicate a novel role for Rho-like GTPases in mitochondrial homeostasis and apoptosis.     

Oxidative stress,NO* and smooth muscle cell extracellular superoxide dismutase expression

Oxygen free radicals apparently play important roles in diseases of the blood vessel wall and increased secretion of superoxide radicals occurs in many situations. The vascular wall contains large amounts of extracellular superoxide dismutase (EC-SOD). The synthesis of the enzyme by the smooth muscle cells (SMC) is modulated by cytokines, growth factors, and vasoactive factors.Here we studied the effects of oxidants (pyrogallol, xanthine oxidase, Cu and Fe), antioxidants (SOD, catalase, and ascorbate), glutathione modulation (n-acetylcysteine and buthionine sulfoximine) and nitric oxide on EC-SOD expression by human vascular SMCs. Generally, the responses in EC-SOD synthesis were small, and no changes were noted in mRNA levels. High concentrations of some of the agents caused reductions in EC-SOD synthesis, mostly concomitantly with toxic effects on the cells. Cell cultures are normally ascorbate deficient, and addition of ascorbate to approach physiological levels doubled the EC-SOD content. Iron ions up-regulated EC-SOD synthesis but also blocked the secretion of the enzyme. Only down-regulation was found by NO*-releasing compounds.In conclusion, there is limited response to oxidant stress of EC-SOD synthesis by SMCs on a cell-autonomous level. The synthesis appears mainly regulated by factors coordinating concerted tissue responses.