Methamphetamine increases Prion Protein and induces dopamine-dependent expression of protease resistant PrPsc.

The cellular prion protein (PrPc) is physiologically expressed within selective brain areas of mammals. Alterations in the secondary structure of this protein lead to scrapie-like prion protein (PrPsc), which precipitates in the cell. PrPsc has been detected in infectious, inherited or sporadic neurodegenerative disorders. Prion protein metabolism is dependent on autophagy and ubiquitin proteasome. Despite not being fully elucidated, the physiological role of prion protein relates to chaperones which rescue cells under stressful conditions.Methamphetamine (METH) is a widely abused drug which produces oxidative stress in various brain areas causing mitochondrial alterations and protein misfolding. These effects produce a compensatory increase of chaperones while clogging cell clearing pathways. In the present study, we explored whether METH administration modifies the amount of PrPc. Since high levels of PrPc when the clearing systems are clogged may lead to its misfolding into PrPsc, we further tested whether METH exposure triggers the appearance of PrPsc. We analysed the effects of METH and dopamine administration in PC12 and striatal cells by using SDS-PAGE Coomassie blue, immune- histochemistry and immune-gold electron microscopy. To analyze whether METH administration produces PrPsc aggregates we used antibodies directed against PrP following exposure to proteinase K or sarkosyl which digest folded PrPc but misfolded PrPsc. We fond that METH triggers PrPsc aggregates in DA-containing cells while METH is not effective in primary striatal neurons which do not produce DA. In the latter cells exogenous DA is needed to trigger PrPsc accumulation similarly to what happens in DA containing cells under the effects of METH. The present findings, while fostering novel molecular mechanisms involving prion proteins, indicate that, cell pathology similar to prion disorders can be mimicked via a DA-dependent mechanism by a drug of abuse.

A subcellular analysis of genetic modulation of PINK1 on mitochondrial alterations, autophagy and cell death.

Mutations in the PTEN-induced putative kinase1 (PINK1) represent the second most frequent cause of autosomal recessive Parkinson's disease. The PINK1 protein mainly localizes to mitochondria and interacts with a variety of proteins, including the pro-autophagy protein beclin1 and the ubiquitin-ligase parkin. Upon stress conditions, PINK1 is known to recruit parkin at the surface of dysfunctional mitochondria and to activate the mitophagy cascade. Aim of this study was to use a simple and highly reproducible catecholamine cell model and transmission electron microscopy to characterize whether PINK1 could affect mitochondrial homeostasis, the recruitment of specific proteins at mitochondria, mitophagy and apoptosis. Samples were analyzed both in baseline conditions and following treatment with methamphetamine (METH), a neurotoxic compound which strongly activates autophagy and produces mitochondrial damage. Our data provide robust sub-cellular evidence that the modulation of PINK1 levels dramatically affects the morphology and number of mitochondria and the amount of cell death. In particular, especially upon METH exposure, PINK1 is able to increase the total number of mitochondria, concurrently recruit beclin1, parkin and ubiquitin and enhance the clearance of damaged mitochondria. In the absence of functional PINK1 and upon autophagy stress, we observe a failure of the autophagy system at large, with marked accumulation of dysfunctional mitochondria and dramatic increase of apoptotic cell death. These findings highlight the strong neuroprotective role of PINK1 as a key protein in the surveillance and regulation of mitochondrial homeostasis.

DNA damage and ubiquitinated neuronal inclusions in the substantia nigra and striatum of mice following MDMA (ecstasy).

RATIONALE: 3,4-Methylenedioxymethamphetamine (MDMA) is an amphetamine derivative, which is neurotoxic to both serotonin (5HT) and dopamine (DA) nerve terminals. Previous reports, carried out in rodents and non-human primates, demonstrated neurotoxicity to monoamine axon terminals, although no study has analyzed nigral and striatal cell bodies at the sub-cellular level. OBJECTIVE: In this study, we examined intrinsic nigral and striatal cells, and PC12 cell cultures to evaluate whether, in mice, MDMA might affect nigral and striatal cell bodies. METHODS: After administering MDMA, we analyzed effects induced in vivo and in vitro using high-performance liquid chromatography (HPLC) analysis, light- and electron microscopy with immunocytochemistry, and DNA comet assay. RESULTS: We found that MDMA (5 mg/kg x4, 2 h apart), besides a decrease of nigrostriatal DA innervation and 5HT loss, produces neuronal inclusions within nigral and intrinsic striatal neurons consisting of multi-layer ubiquitin-positive whorls extending to the nucleus of the cell. These fine morphological changes are associated with clustering of heat shock protein (HSP)-70 in the nucleus, very close to chromatin filaments. In the same experimental conditions, we could detect oxidation of DNA bases followed by DNA damage. The nature of inclusions was further investigated using PC12 cell cultures. CONCLUSIONS: The present findings lead to re-consideration of the neurotoxic consequences of MDMA administration. In fact, occurrence of ubiquitin-positive neuronal inclusions and DNA damage both in nigral and striatal cells sheds new light into the fine alterations induced by MDMA, also suggesting the involvement of nuclear and cytoplasmic components of the ubiquitin-proteasome pathway in MDMA toxicity.

A3 adenosine receptors in human astrocytoma cells: agonist-mediated desensitization, internalization, and down-regulation.

A(3) adenosine receptor activation has been previously demonstrated to result in both neuroprotective and neurodegenerative effects, depending upon specific pathophysiological conditions. This dual effect may depend on receptor regulation mechanisms that are able to change receptor availability and/or function. In the present study, we investigated desensitization, internalization, and down-regulation of native A(3) adenosine receptors in human astrocytoma cells after exposure to the agonist 2-chloro-N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (Cl-IBMECA). Cl-IBMECA induced a concentration-dependent inhibition of adenylyl cyclase activity with an EC(50) value of 2.9 +/- 0.1 nM. The effect was suggested to be mediated by A(3) adenosine receptor subtype by the use of selective adenosine receptor antagonists. Cell treatment with pertussis toxin abolished Cl-IBMECA-mediated inhibition of adenylyl cyclase activity, evidencing an A(3) receptor coupling to inhibitory G protein. Short-term exposure to the agonist Cl-IBMECA (100 nM) caused rapid receptor desensitization, within 15 min. Agonist-induced desensitization was accompanied by receptor internalization: A(3) adenosine receptor internalized with rapid kinetics, within 30 min, after cell exposure to 100 nM Cl-IBMECA. The localization of A(3) adenosine receptors on the plasma membrane and in intracellular compartments was directly revealed by immunogold electron microscopy. After desensitization, the removal of agonist led to the restoration of A(3) adenosine receptor functioning through receptor recycling to the cell surface within 120 min. Prolonged agonist exposure (1-24 h) resulted in a marked down-regulation of A(3) adenosine receptors that reached 21.9 +/- 2.88% of control value after 24 h. After down-regulation, the recovery of receptor functioning was slow (24 h) and associated with the restoration of receptor levels close to control values. In conclusion, our results demonstrated that A(3) receptors, in astrocytoma cells, are regulated after short- and long-term agonist exposure.

Yolk utilization in stick insects entails the release of vitellin polypeptides into the perivitelline fluid.

This study investigates the developmental fate of vitellin (Vt) polypeptides generated by limited proteolysis in an insect embryo. To this end, a number of polyclonal (pAb) and monoclonal antibodies (mAb) were raised against the yolk sac and the perivitelline fluid of late embryos of the stick insect Carausius morosus. Two dimensional immuno gel electrophoresis and Western blotting demonstrate that polypeptides resulting from Vt processing are present both in the yolk sac and the perivitelline fluid. At the confocal microscope, different labelling patterns were detected in the ooplasm depending on the stage of development attained by the embryo. At early developmental stages, label is associated with large unsegmented portions of the fluid ooplasm. During embryonic development, the fluid ooplasm is gradually transformed into yolk granules by intervention of vitellophages. Prior to dorsal closure, the yolk sac is separated from the perivitelline fluid by interposition of serosa cells (the so called serosa membrane). Several mAbs raised against the perivitelline fluid react specifically with this membrane suggesting that the release of Vt polypeptides from the yolk sac occurs by intracellular transit through the serosa cells. By immunocytochemistry, gold label appears associated with the cell surface and a number of vacuoles of the serosa membrane. These data are interpreted as suggesting that Vt polypeptides resulting from limited proteolysis in stick insect embryos are not exhaustively degraded within the yolk sac, but are instead transferred transcytotically to the perivitelline fluid through the serosa membrane.

Agonist-induced internalization and recycling of the human A(3) adenosine receptors: role in receptor desensitization and resensitization.

A(3) adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen-dependent nature of the therapeutic effects probably related to receptor desensitization and down-regulation. Here we studied the agonist-induced internalization of human A(3) adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N(6)-(4-amino-3-[(125)I]iodobenzyl)adenosine-5'-N-methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A(3) adenosine receptor showed a profile typical of these receptors in other cell lines (K:(D) = 1.3+/-0.08 nM; B(max) = 400+/-28 fmol/mg of proteins). The iodinated agonist, bound at 4 degrees C to whole transfected cells, was internalized by increasing the temperature to 37 degrees C with a rate constant of 0.04+/-0.034 min(-1). Agonist-induced internalization of A(3) adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short-term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02+/-0.0017 min(-1). Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.

A(1) adenosine receptors in human neutrophils: direct binding and electron microscope visualization.

By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical studies have shown that A(1) adenosine receptors modulate chemotaxis in response to chemotactic peptides. Until now, the characteristics of the specific agonist binding and the visualization of A(1) receptors in human neutrophils have not been investigated. In the present study, we used the agonist [(3)H] CHA for radioligand binding studies and a CHA-biotin XX probe in order to visualize the A(1) binding sites in human neutrophils, ultrastructurally, by conjugation with colloidal gold-streptavidin. [(3)H] CHA bound A(1) adenosine receptors with selectivity and specificity, although with a low binding capacity. Scatchard analysis showed a Kd value of 1.4 +/- 0.08 nM and a maximum density of binding sites of 7.1 +/- 0.37 fmol/mg of proteins. The good affinity and selectivity of the CHA-biotin XX probe for A(1) adenosine receptors allowed us to visualize them, after conjugation with colloidal gold-streptavidin, as electron-dense gold particles on the neutrophil surface and inside the cell. The internalization of the ligand-receptor complex was followed in a controlled temperature system, and occurred through a receptor-mediated pathway. The kinetics of the intracellular trafficking was fast, taking less than 5 min. These data suggest that the CHA-biotin XX-streptavidin-gold complex is a useful marker for the specific labelling of A(1) binding sites and to follow the intracellular trafficking of these receptors.

[Correlation of ultrasound and galactography in the diagnosis of nipple discharge. Preliminary results]. / Correlazione tra ecografia e galattografia nella diagnostica della mammella secernente. Risultati preliminari.

INTRODUCTION: Several pathologic conditions involving the breast ductal tree can cause bloody or serous nipple discharge. Galactography plays a major clinical role in identifying and localizing intraductal masses, but its sensitivity in detecting cancer is certainly suboptimal. Presently high-frequency ultrasound (US) probes allow detection and guided biopsy of intraductal lesions. We compared the specific information provided by US and galactography in the discharging breast. MATERIAL AND METHODS: Thirty-three patients with discharging breast were submitted to both diagnostic examinations. US was performed with 13 MHz scanheads both before and after galactography. Galactography was performed with 30-31 G catheters to cannulate the discharging duct. Nonionic, water-soluble, sterile contrast material was administered. Postgalactography US was performed to investigate if it could yield further information. The final diagnosis was made at histology and 2 years' instrumental follow-up. RESULTS: Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were evaluated for both techniques. We considered a positive finding the detection of a lesion in general (be it papilloma, papillomatosis, or cancer), as well as the detection of carcinoma only. Sensitivity was 96% for galactography and 84% for US in the former case, versus 50% and 100%, respectively, in the latter. Postgalactography US added no major information. DISCUSSION AND CONCLUSION: US is more sensitive than galactography in cancer diagnosis and, it permits guided biopsy and preoperative localization of unpalpable ductal lesions. In our limited experience, US can be considered a complementary diagnostic tool to galactography in the discharging breast.

A fat body derived protein is selectively sulfated in the stick insect ovary by transcytosis through the follicular epithelium.

With the onset of vitellogenesis, the follicular epithelium overlying the oocyte in stick insect ovarioles becomes highly polarized and patent by formation of wide intercellular spaces. The aim of the present study was to provide experimental support to the notion that the follicular epithelium in this insect species may be involved in transcytosis. Data demonstrate that the follicular epithelium carries out sulfo-conjugation of a 85 kDa fat body derived protein by allowing it ot transit from one cell pole to another. Along the basal end, follicle cells branch into a number of cytoplasmic finger-like projections. At the opposite end facing the oocyte they taper off into lance-head shapes. Different vesicular elements are evident at both these extremities. In vivo exposure to horseradish peroxidase shows that the vesicular elements present along the apical end provide an endocytic entry. In contrast, those present along the basal end are labeled with sodium [35S]-sulfate, suggesting that they may be exocytic vesicles containing a sulfo-conjugated secretory product. In vivo exposure to sodium [35S]-sulfate caused radioactivity to appear over the Golgi apparatus and some nearby vesicles of the follicle cell cytoplasm, including the exocytic vesicles. The intracellular pathway of the follicle cells was also examined by immunogold labeling using a monoclonal antibody raised against a 85 kDa fat body derived protein. Under these conditions, gold particles were consistently detected over the Golgi apparatus and the vesicular elements lying along both poles of the follicle cell membrane. Based on this evidence, it is concluded that follicular cells in stick insect ovarioles are endowed with the ability to undergo transcytosis by providing an endocytic entry along the apical end and by releasing exocytically a sulfo-conjugated 85 kDa protein along the baso-lateral domain of the follicle cell membrane.

Vitellogenin is glycosylated in the fat body of the stick insect Carausius morosus and not further modified upon transfer to the ovarian follicle.

Synthesis and secretion of vitellogenin (Vg) polypeptides were studied in egg-laying females of the stick insect Carausius morosus following in vivo exposure to [35S]-methionine and acetyl-N-[3H]-glucosamine. The specificity of radioisotope incorporation was assessed by in vitro inhibition with tunicamycin and carbohydrate extraction with endo-glycosidase H. Vg polypeptides change in molecular weight during synthesis in the fat body and are not further modified upon transfer to the haemolymph or to the oocyte, suggesting that they are already fully glycosylated prior to secretion. Radioactivity in the fat body was initially distributed over cisternae of the rough endoplasmic reticulum and gradually transferred to the Golgi apparatus. Within an hour of exposure, electron-dense granules budding from the trans-Golgi network became preferentially labeled. Radioactivity in the ovarian follicle was restricted to the yolk granules of the cortical ooplasm and to the amorphous material lying within the intercellular channels of the follicular epithelium. This amorphous material was also shown to react positively when tested with a monoclonal antibody raised specifically against a Vg polypeptide.

Oogenesis in Actinoposthia beklemischevi (Platyhelminthes, Acoela): an ultrastructural and cytochemical study.

The oogenesis of the acoel Actinoposthia beklemischevi can be divided into a previtellogenic and a vitellogenic stage. Maturing oocytes are surrounded by accessory cells (a.c.) that produce electrondense granules, the content of which is released into the space between the oocyte and a.c. and gives rise to a thin primary egg envelope. The a.c. may also contribute to yolk synthesis by transferring low molecular weight precursors to the oocyte. Two types of inclusion are produced in maturing oocytes. Type I inclusions are small, roundish granules produced by the Golgi complex. They have a proteinaceous non-polyphenolic content which is discharged in the intercellular space and produce a thicker secondary egg envelope. Type I inclusions represent eggshell-forming granules (EFGs). Type II inclusions are variably sized globules progressively changing their shape from round to crescent. They appear to be produced by the ER, contain glycoproteins and remain scattered throughout the cytoplasm in large oocytes. Type II inclusions represent yolk. The main features of oogenesis in Actinoposthia are: (a) EFGs have a non-polyphenolic composition; (b) the egg envelope has a double origin and is not sclerotinized; (c) yolk production appears to be autosynthetic. The present ultrastructural findings are compared with those from other Acoelomorpha and Turbellaria.

Ultrastructural and cytochemical aspects of oogenesis in Castrada viridis (Platyhelminthes, Rhabdocoela).

The heterocellular female gonad of the typhloplanoid Castrada viridis consists of a single germarium and two rows of vitellaria. The germarium, composed of a germinative zone and a growth zone, is surrounded externally by a layer of accessory cells the function of which is hypothesized in this study. The main feature of oocyte differentiation is the synthesis of small electron-dense inclusions produced by the rough endoplasmic reticulum (R.E.R.) and Golgi complex. The electron-dense content of the egg inclusions reacts positively to the cytochemical test used to detect polyphenols and is only partially extracted following incubation in protease. The genesis, composition, and peripheral location of egg inclusions in mature oocytes suggest that they could represent residual eggshell granules. The presumed function of eggshell granules is discussed and their fine morphology is compared with that observed in other neoophoran Platyhelminthes.