The Role of Vesicle Trafficking and Release in Oligodendrocyte Biology

Oligodendrocytes are a subtype of glial cells found within the central nervous system (CNS), responsible for the formation and maintenance of specialized myelin membranes which wrap neuronal axons. The development of myelin requires tight coordination for the cell to deliver lipid and protein building blocks to specific myelin segments at the right time. Both internal and external cues control myelination, thus the reception of these signals also requires precise regulation. In late years, a growing body of evidence indicates that oligodendrocytes, like many other cell types, may use extracellular vesicles (EVs) as a medium for transferring information. The field of EV research has expanded rapidly over the past decade, with new contributions that suggest EVs might have direct involvement in communications with neurons and other glial cells to fine tune oligodendroglial function. This functional role of EVs might also be maladaptive, as it has likewise been implicated in the spreading of toxic molecules within the brain during disease. In this review we will discuss the field’s current understanding of extracellular vesicle biology within oligodendrocytes, and their contribution to physiologic and pathologic conditions.

     

Dominant-negative effects in prion diseases: insights from molecular dynamics simulations on mouse prion protein chimeras

Mutations in the prion protein (PrP) can cause spontaneous prion diseases in humans (Hu) and animals. In transgenic mice, mutations can determine the susceptibility to the infection of different prion strains. Some of these mutations also show a dominant-negative effect, thus halting the replication process by which wild type mouse (Mo) PrP is converted into Mo scrapie. Using all-atom molecular dynamics (MD) simulations, here we studied the structure of HuPrP, MoPrP, 10?Hu/MoPrP chimeras, and 1 Mo/sheepPrP chimera in explicit solvent. Overall, ～2?μs of MD were collected. Our findings suggest that the interactions between α1 helix and N-terminal of α3 helix are critical in prion propagation, whereas the β2–α2 loop conformation plays a role in the dominant-negative effect.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:4.     

RET oncoproteins induce tyrosine phosphorylation changes of proteins involved in RNA metabolism

We report the identification of proteins induced in response to RET/PTC2, an oncogene implicated in thyroid cancers. Anti-phosphotyrosine antibody affinity resin was used to purify Tyr(P)-containing and interacting proteins from 293T and NIH3T3 cells which were transfected with kinase active or inactive RET/PTC and RETMEN2 oncogenes. Proteins were separated by one-dimensional SDS-PAGE, extracted by in-gel digestion, and identified by MALDI-TOF peptide mass fingerprinting. The expression and tyrosine phosphorylation of Sam68, a protein implicated in mRNA nucleocytoplasmic translocation and splicing, were further examined in RET-transfected cells and thyroid tumors. Of relevance, cells transfected with RETMEN2B examined for anti-phosphotyrosine bound proteins, showed other proteins implicated in splicing: DEAD-box p68 RNA helicase, SYNCRIP, and hnRNP K. Western blotting analysis suggested that these proteins are singularly tyrosine phosphorylated in RETMEN2B-transfected cells, and that they constitutively bind with Sam68. The study concludes that regulation of splicing factors is likely to be important in RET-mediated thyroid carcinogenesis.     

Body Mass Index and Serum Proteomic Profile in Breast Cancer and Healthy Women: A Prospective Study

Epidemiological studies suggest a possible association between BMI, diagnosis and clinical-pathological breast cancer characteristics but biological bases for this relationship still remain to be ascertained. Several biological mechanisms play a role in the genesis and progression of breast cancer. This study aimed to investigate relationships between BMI and breast cancer diagnosis/progression in a Southern Italian population and to try to interpret results according to the serum proteomic profile of healthy and breast cancer patients.BMI, presence or absence of breast cancer and its clinical-pathological characteristics were analyzed in a series of 300 breast cancer women and compared with those of 300 healthy women prospectively. To investigate whether obesity is associated with alterations in serum protein profile, SELDI-ToF approach was applied.Alcohol consumption (22.7% vs 11.3%; p<0.001) and postmenopausal status (65.7% vs 52%; p<0.001) but not BMI resulted significantly different in patients vs controls. Conversely, BMI was significantly associated with a larger-tumour size (BMI> = 30 respect to normal weight: OR = 2.49, 95% CI 1.25–4.99, p = 0.0098) and a higher probability of having positive axillary lymph node (OR = 3.67, CI 95% 2.16–6.23, p<0.0001). Multivariate analysis confirmed the association of breast cancer diagnosis with alcohol consumption (OR = 2.28;CI 1.36–3.83; p<0.0018). Serum protein profile revealed the presence of significant (p-value <0,01) differentially expressed peaks m/z 6934, m/z 5066 in high BMI breast cancer patients vs healthy subjects and m/z 6934, m/z 3346 in high vs low BMI breast cancer patients.The analysis of pathological features of cancer indicates that normal weight women have a significantly higher probability of having a smaller breast cancer at time of diagnosis and negative axillary lymph nodes while increased BMI is associated with an altered protein profile in breast cancer patients. Further studies to identify specific proteins found in the serum and their role in breast cancerogenesis and progression are in progress.     

Design and optimization of lentiviral vectors for transfer of GALC expression in Twitcher brain

BACKGROUND: Demyelination in globoid cell leukodystrophy (GLD) is due to a deficiency of galactocerebrosidase (GALC) activity. Up to now, in vivo brain viral gene transfer of GALC showed modest impact on disease development in Twitcher mice, an animal model for GLD. Lentiviral vectors, which are highly efficient to transfer the expression of therapeutic genes in neurons and glial cells, have not been evaluated for direct cerebral therapy in GLD mice. METHODS: Lentiviral vectors containing the untagged cDNA or the hemagglutinin (HA)-tagged cDNA for the full-length mouse GALC sequence were generated and validated in vitro. In vivo therapeutic efficacy of these vectors was evaluated by histology, biochemistry and electrophysiology after transduction of ependymal or subependymal layers in young Twitcher pups. RESULTS: Both GALC lentiviral vectors transduced neurons, oligodendrocytes and astrocytes with efficiencies above 75% and conferred high levels of enzyme activity. GALC accumulated in lysosomes of transduced cells and was also secreted to the extracellular medium. Conditioned GALC medium was able to correct the enzyme deficiency when added to non-transduced Twitcher glial cultures. Mice that received intraventricular injections of GALC vector showed accumulation of GALC in ependymal cells but no diffusion of the enzyme from the ependymal ventricular tree into the cerebral parenchyma. Significant expression of GALC-HA was detected in neuroglioblasts when GALC-HA lentiviral vectors were injected in the subventricular zone of Twitcher mice. Life span and motor conduction in both groups of treated Twitcher mice were not significantly ameliorated. CONCLUSIONS: Lentiviral vectors showed to be efficient for reconstitution of the GALC expression in Twitcher neural cells. GALC was able to accumulate in lysosomes as well as to enter the secretory pathway of lysosomal enzymes, two fundamental aspects for gene therapy of lysosomal storage diseases. Our in vivo results, while showing the capacity of lentiviral vectors to transfer expression of therapeutic GALC in the Twitcher brain, did not limit progression of disease in Twitchers and highlight the need to evaluate other routes of administration.     

Psychosine,the cytotoxic sphingolipid that accumulates in globoid cell leukodystrophy,alters membrane architecture

Globoid cell leukodystrophy (GLD) is a neurological disease caused by deficiency of the lysosomal enzyme galactosylceramidase (GALC). In the absence of GALC, the cytotoxic glycosphingolipid, psychosine (psy), accumulates in the nervous system. Psychosine accumulation preferentially affects oligodendrocytes, leading to progressive demyelination and infiltration of activated monocytes/macrophages into the CNS. GLD is characterized by motor defects, cognitive deficits, seizures, and death by 2–5 years of age. It has been hypothesized that psychosine accumulation, primarily within lipid rafts, results in the pathogenic cascade in GLD. However, the mechanism of psychosine toxicity has yet to be elucidated. Therefore, we synthesized the enantiomer of psychosine (ent-psy) to use as a probe to distinguish between protein-psy (stereo-specific enantioselective) or membrane-psy (stereo-insensitive nonenantioselective) interactions. The enantiomer of psychosine has equal or greater toxicity compared with psy, suggesting that psy exerts its toxicity through a nonenantioselective mechanism. Finally, in this study we demonstrate that psy and ent-psy localize to lipid rafts, perturb natural and artificial membrane integrity, and inhibit protein Kinase C translocation to the plasma membrane. Although other mechanisms may play a role in disease, these data strongly suggest that psy exerts its effects primarily through membrane perturbation rather than through specific protein-psy interactions.     

Increased susceptibility to degradation by trypsin and subtilisin of in vitro peroxidized myelin proteins

We examined the possibility that the peroxidative damage to central nervous system myelin produced by reactive oxygen species (ROS), could modify the susceptibility of its proteins to the proteolytic action of proteases such as trypsin and subtilisin. Purified myelin membranes obtained from adult rat brains were in vitro peroxidized by two non-enzymatic systems: Fe³⁺ plus ascorbic acid and Cu²⁺ plus hydrogen peroxide. Myelin proteins were severely affected by peroxidation. There was an increase in the amount of carbonyl groups (CO), accompanied by and enhanced susceptibility to degradation by trypsin and subtilisin of myelin basic proteins (MBP) and of the major proteolipid protein (PLP). The effect upon the degradation of myelin protein is a possible consequence of the appearance in the structure of myelin proteins of peroxidative modifications that contribute to the recognition by proteolytic enzymes. This hypothesis is supported by the fact that if peroxidation of myelin membranes is done in the presence of EDTA, both CO formation and increased sensitivity to enzymatic breakdown disappear. These results suggest that the appearance of abnormal post-translational modifications in the myelin membrane produced by peroxidation could constitute a putative mechanism of modulating the capacity of myelin proteins to be metabolized by proteases.     

A novel activating mutation in the RET tyrosine kinase domain mediates neoplastic transformation

We report the finding of a novel missense mutation at codon 833 in the tyrosine kinase of the RET proto-oncogene in a patient with a carcinoma of the thyroid. In vitro experiments demonstrate that the R833C mutation induces transformed foci only when present in the long 3' splice isoform and, in keeping with a model in which the receptor has to dimerize to be completely activated, glial cell line-derived neurotrophic factor stimulation leads the RET(R833C) receptor to a higher level of activation. Tyrosine kinase assays show that the RET(R833C) long isoform has weak intrinsic kinase activity and phosphorylation of an exogenous substrate is not elevated even in the presence of glial cell line-derived neurotrophic factor. Furthermore, the R833C mutation is capable of sustaining the transformed phenotype in vivo but does not confer upon the transformed cells the ability to degrade the basement membrane in a manner analogous to metastasis. Our functional characterization of the R833C substitution suggests that, like the V804M and S891A mutations, this tyrosine kinase mutation confers a weak activating potential upon RET. This is the first report demonstrating that the introduction of an intracellular cysteine can activate RET. However, this does not occur via dimerization in a manner analogous to the extracellular cysteine mutants.