Glial Fibrillary Acidic Protein is Greatly Modified by Oxidative Stress in Aceruloplasminemia Brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

IGF-1 promotes β-amyloid production by a secretase-independent mechanism

β-Amyloid peptide (Aβ) is generated via the sequential proteolysis of β-amyloid precursor protein (APP) by β- and γ-secretases, and plays a crucial role in the pathogenesis of Alzheimer’s disease (AD). Here, we sought to clarify the role of insulin-like growth factor-1 (IGF-1), implicated in the AD pathomechanism, in the generation of Aβ. Treatment of neuroblastoma SH-SY5Y cells expressing AD-associated Swedish mutant APP with IGF-1 did not alter cellular levels of APP, but significantly increased those of β-C-terminal fragment (β-CTF) and secreted Aβ. IGF-1 also enhanced APP phosphorylation at Thr668. Treatment of β-CTF-expressing cells with IGF-1 increased the levels of β-CTF and secreted Aβ. The IGF-1-induced augmentation of β-CTF was observed in the presence of γ-secretase inhibitors, but not in cells expressing β-CTF with a Thr668 to alanine substitution. These results suggest that IGF-1 promotes Aβ production through a secretase-independent mechanism involving APP phosphorylation.     

Cell-cycle-dependent Xenopus TRF1 recruitment to telomere chromatin regulated by Polo-like kinase

Telomeres are regulated by a homeostatic mechanism that includes telomerase and telomeric repeat binding proteins, TRF1 and TRF2. Recently, it has been hypothesized that telomeres assume distinct configurations in a cell-cycle-dependent manner, although direct biochemical evidence is lacking. Here we demonstrated that Xenopus TRF1 (xTRF1) associates with telomere chromatin specifically in mitotic Xenopus egg extracts, and dissociates from it upon mitotic exit. Both the N-terminal TRF-homology (TRFH) domain and the linker region connecting the TRFH domain and the C-terminal Myb domain are required for this cell-cycle-dependent association of xTRF1 with chromatin. In contrast, Xenopus TRF2 (xTRF2) associates with chromatin throughout the cell cycle. We showed that Polo-like kinase (Plx1) phosphorylates xTRF1 in vitro. Moreover, the mitotic xTRF1-chromatin association was significantly impaired when Plx1 was immunodepleted from the extracts. Finally, high telomerase activities were detected in association with replicating interphase chromatin compared with mitotic chromatin. These results indicate that telomere chromatin is actively regulated by cell-cycle-dependent processes, and provide an insight for understanding how telomeres undergo DNA metabolisms during the cell cycle.     

Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P. falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite.     

Spatial control of branching within dendritic arbors by dynein-dependent transport of Rab5-endosomes

Dendrites allow neurons to integrate sensory or synaptic inputs, and the spatial disposition and local density of branches within the dendritic arbor limit the number and type of inputs. Drosophila melanogaster dendritic arborization (da) neurons provide a model system to study the genetic programs underlying such geometry in vivo. Here we report that mutations of motor-protein genes, including a dynein subunit gene (dlic) and kinesin heavy chain (khc), caused not only downsizing of the overall arbor, but also a marked shift of branching activity to the proximal area within the arbor. This phenotype was suppressed when dominant-negative Rab5 was expressed in the mutant neurons, which deposited early endosomes in the cell body. We also showed that 1) in dendritic branches of the wild-type neurons, Rab5-containing early endosomes were dynamically transported and 2) when Rab5 function alone was abrogated, terminal branches were almost totally deleted. These results reveal an important link between microtubule motors and endosomes in dendrite morphogenesis.     

Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex

Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project.     

Nanoscopic lipid domain dynamics revealed by atomic force microscopy

Intrinsic heterogeneities, represented as domain formations in biological membranes, are important to both the structure and function of the membranes. We observed domain formations in mixed lipid bilayers of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol) in a fluid environment using an atomic force microscope (AFM). At room temperature, we demonstrated that both microscopic and nanoscopic domains coexist and the DPPC-rich domain is approximately 1.4 nm higher than the surrounding DLPC-rich membrane areas as a consequence of intrinsic phase differences. DPPC-rich microscopic domains became larger as DPPC concentration increased. In cholesterol-free mixtures, nanoscopic DPPC-rich domain sizes ranged from 26 to 46 nm depending on phospholipid concentration. Domain size varied between 33 and 48 nm in the presence of cholesterol (0 < or = [chol] < or = 40). The nanoscopic domains were markedly fragmented near [chol] = 0.135 and appeared to fuse more readily into microscopic domains at higher and lower [chol]. By phase balance analyses we demonstrated phase behavior differences between a free-vesicle GUV system studied by confocal light microscopy and a supported membrane system studied by AFM. We propose a new three-dimensional phase diagram elucidating the effects of a solid substrate support on lipid phase behavior relevant to complex membrane phase phenomena in biological systems.     

Telomeric DNA ends are essential for the localization of Ku at telomeres in fission yeast

The Ku70-Ku80 heterodimer is a conserved protein complex essential for the non-homologous end-joining pathway. Ku proteins are also involved in telomere maintenance, although their precise roles remain to be elucidated. In fission yeast, pku70(+), the gene encoding the Ku70 homologue, has been reported. Here we report the identification and characterization of pku80(+), the gene encoding Ku80. Both pku70(+) and pku80(+) are essential for efficient non-homologous end-joining. We also found that the pku70 and pku80 mutants are sensitive to methyl methanesulfonate and hydroxyurea, suggesting their roles in the S phase. The pku80 mutant shows telomere shortening and tandem amplification of a subtelomeric sequence but no defects in the telomere position effect, as was previously reported for the pku70 mutant. By using the chromatin immunoprecipitation assay, we demonstrated that Pku70 and Pku80 physically interact with telomeric repeats and subtelomeric sequences. Interestingly, this telomere association of Pku proteins is independent of Taz1, a telomeric DNA-binding protein. We also showed that the Pku proteins do not associate with ectopically integrated telomeric repeats in the internal region of circular chromosomes. These results indicate that the physical end of DNA is necessary for the localization of Pku80 at telomeres.