An examination of current Hg deposition and export in Fenno-Scandian catchments

The input and output flux data of total Hg (THg) and methylmercury (MeHg) from three catchments located in different geographical regions in Sweden and one catchment in southern Finland were compared to elucidate the role of current atmospheric Hg/MeHg deposition with regard to waterborne Hg/MeHg output.There was a negative co-variaton between the open field THg inputs and the ratio of THg output to open field input. The highest ratio (and lowest input) occurring in N. Sweden and S. Finland, while the lowest output ratio (and highest inputs) occurred in southwest Sweden. A much larger variation was found in the ratio of output to open field input for MeHg (14 to 160%). Examinations of MeHg input/output data in relation to catchment charateristics suggest that riparian peat, mires and wet organic soil contributed to the large MeHg output from certain catchments, probably due to in situ production of MeHg. This finding is consistent with other studies which have found that catchment characteristics such as wetland area, flow pathways, seasonal temperature and water flow are important in controlling the output of MeHg. These catchment characteristics govern the fate of the contemporary input of Hg and MeHg as well as the mobilization of the soil pools.     

O-GlcNAcylation increases non-amyloidogenic processing of the amyloid-β precursor protein (APP)

The amyloid-β precursor protein (APP) was shown to be O-GlcNAcylated 15 years ago, but the effect of this modification on APP processing and formation of the Alzheimer’s disease associated amyloid-β (Aβ) peptide has so far not been investigated. Here, we demonstrate with pharmacological tools or siRNA that O-GlcNAcase and O-GlcNAc transferase regulate the level of O-GlcNAcylated APP. We also show that O-GlcNAcylation increases non-amyloidogenic α-secretase processing, resulting in increased levels of the neuroprotective sAPPα fragment and decreased Aβ secretion. Our results implicate O-GlcNAcylation as a potential therapeutic target for Alzheimer’s disease.     

Noninvasive Monitoring of Apoptosis versus Necrosis in a Neuroblastoma Cell Line Expressing a Nuclear Pore Protein Tagged with the Green Fluorescent Protein

A fusion chimera between the integral nuclear pore membrane protein POM121 and GFP (green fluorescent protein) has been shown to correctly target to the nuclear pores when transiently expressed in a number of mammalian cell types. POM121-GFP is therefore an excellent marker for the noninvasive studies of the nuclear pores in living cells using fluorescence microscopy. We have established a line of neuroblastoma cells stably expressing the POM121-GFP fusion protein. We also monitored the nuclear envelope in living cells after induction of apoptosis or necrosis using 1 μM staurosporine or 100 μMp-benzoquinone, respectively. Interestingly, the POM121-GFP fluorescence was weaker or missing in the apoptotic cells. The disappearance of the nuclear pore marker accompanied apoptotic progression as judged by the degree of chromatin condensation and DNA fragmentation as analyzed by DNA staining and TUNEL assay, respectively. In contrast, the intensity of the nuclear rim fluorescence was unaffected in necrotic cells displaying an abnormal morphology with tilted nuclei. Thus, it was possible to distinguish between apoptotic and necrotic development in living cells using fluorescence microscopy. This cell line provides a fast and convenient model for screening suspected toxic xenobiotics.     

PI3-K- and PKC-dependent up-regulation of APP processing enzymes by retinoic acid

Retinoic acid stimulates α-secretase processing of amyloid precursor protein (APP) and decreases β-secretase cleavage that leads to amyloid-β formation. Here, we investigated the effect of retinoic acid on the two putative α-secretases, the disintegrin metalloproteinases ADAM10 and TACE, and the β-site cleaving enzyme BACE1, in human neuroblastoma SH-SY5Y cells. Western blot analysis showed that exposure to retinoic acid resulted in significantly increased levels of ADAM10 and TACE, suggesting that regulation of α-secretases causes the effects on APP processing. The presence of the phosphatidylinositol 3-kinase inhibitor LY 294002 selectively reduced the effect on ADAM10 protein levels but not on ADAM10 mRNA levels as determined by RT-PCR. On the other hand, the effect on TACE was shown to be dependent on protein kinase C, since it was completely blocked in the presence of the inhibitor bisindolylmaleimide XI. Our data indicate that different signalling pathways are involved in retinoic acid-induced up-regulation of the secretases.     

IGF-1-induced processing of the amyloid precursor protein family is mediated by different signaling pathways

The mammalian amyloid precursor protein (APP) protein family consists of the APP and the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). The neurotoxic amyloid beta-peptide (Abeta) originates from APP, which is the only member of this protein family implicated in Alzheimer disease. However, the three homologous proteins have been proposed to be processed in similar ways and to have essential and overlapping functions. Therefore, it is also important to take into account the effects on the processing and function of the APP-like proteins in the development of therapeutic drugs aimed at decreasing the production of Abeta. Insulin and insulin-like growth factor-1 (IGF-1) have been shown to regulate APP processing and the levels of Abeta in the brain. In the present study, we show that IGF-1 increases alpha-secretase processing of endogenous APP and also increases ectodomain shedding of APLP1 and APLP2 in human SH-SY5Y neuroblastoma cells. We also investigated the role of different IGF-1-induced signaling pathways, using specific inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK). Our results indicate that phosphatidylinositol 3-kinase is involved in ectodomain shedding of APP and APLP1, but not APLP2, and that MAPK is involved only in the ectodomain shedding of APLP1.     

Correlation between nucleocytoplasmic transport and caspase-3-dependent dismantling of nuclear pores during apoptosis

During apoptosis (also called programmed cell death), the chromatin condenses and the DNA is cleaved into oligonucleosomal fragments. Caspases are believed to play a major role in nuclear apoptosis. However, the relation between dismantling of nuclear pores, disruption of the nucleocytoplasmic barrier, and nuclear entry of caspases is unclear. We have analyzed nuclear import of the green fluorescent protein fused to a nuclear localization signal (GFP-NLS) in tissue culture cells undergoing apoptosis. Decreased nuclear accumulation of GFP-NLS could be detected at the onset of nuclear apoptosis manifested as dramatic condensation and redistribution of chromatin toward the nuclear periphery. At this step, dismantling of nuclear pores was already evident as indicated by proteolysis of the nuclear pore membrane protein POM121. Thus, disruption of nuclear compartmentalization correlated with early signs of nuclear pore damage. Both these events clearly preceded massive DNA fragmentation, detected by TUNEL assay. Furthermore, we show that in apoptotic cells, POM121 is specifically cleaved at aspartate-531 in its large C-terminal portion by a caspase-3-dependent mechanism. Cleavage of the C-terminal portion of POM121, which is adjoining the nuclear pore complex, is likely to disrupt interactions with other nuclear pore proteins affecting the stability of the pore complex. A temporal correlation of apoptotic events supports a model where caspase-dependent disassembly of nuclear pores and disruption of the nucleocytoplasmic barrier paves the way for nuclear entry of caspases and subsequent activation of CAD-mediated DNA fragmentation.     

Increased processing of APLP2 and APP with concomitant formation of APP intracellular domains in BDNF and retinoic acid-differentiated human neuroblastoma cells

The amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. We have previously shown that all members of the APP protein family are up-regulated upon retinoic acid (RA)-induced neuronal differentiation of SH-SY5Y neuroblastoma cells. Here, we demonstrate that RA also affects the processing of APLP2 and APP, as shown by increased shedding of both sAPLP2 and sAPPalpha, as well as elevated levels of the APP intracellular domains (AICDs). Brain-derived neurotrophic factor (BDNF) has been reported to induce APP promoter activity and RA induces expression of the tyrosine kinase receptor B (TrkB) in neuroblastoma cells. We show that the increase in shedding of both APLP2 and APP in response to RA is not mediated through the TrkB receptor. However, BDNF concomitant with RA increased the expression of APP even further. In addition, the secretion of sAPLP2 and sAPPalpha as well as the levels of AICDs were increased in response to BDNF. In contrast, the levels of membrane-bound APP C-terminal fragment C99 significantly decreased. Our results suggest that RA and BDNF shifts APP processing towards the alpha-secretase pathway. In addition, we show that RA and BDNF regulate N-linked glycosylation of APLP1.     

Acute and chronic studies on functional aspects of coexistence

Autoinhibition of acetylcholine release by the coexisting peptide galanin in the septal afferents to the hippocampus of the rat was examined in tissue slices from the hippocampus. Galanin inhibits the evoked release of the coexisting neurotransmitter, acetylcholine, in the ventral hippocampus, providing an example of autoinhibition of release of a neurotransmitter by one of the coexisting neurotransmitters. The galanin mediated inhibition of the acetylcholine release is a complement to the well known strong cholinergic autoinhibition. The effects of the coexisting galanin and acetylcholine on several second messenger systems were also examined: acetylcholine acting at muscarinic receptors depresses cyclic adenosine 3',5'-monophosphate and stimulates elevation of cyclic guanosine 3',5'-monophosphate levels, whereas neither cyclic adenosine 3',5'-monophosphate nor cyclic guanosine 3',5'-monophosphate levels were affected by galanin (1 microM). Galanin however inhibited partly the muscarinic stimulation of phosphoinositide breakdown, suggesting that inositol phosphate(s) or diacylglycerol may act as second messenger(s) of the galanin action in the hippocampus. The effects of chronic changes in firing rate on the coexisting neurotransmitters in the rat ventral spinal cord containing serotonin, thyrotropin releasing hormone, substance P and substance K were examined. The tissue levels of the coexisting transmitters were studied in rats chronically treated with imipramine (14 days; 2 x 10 mumoles/kg/day) and zimelidine (14 days; 2 x 10 mumoles/kg/day). Upon treatment with zimelidine the tissue levels of the serotonin metabolite 5-hydroxyindoleacetic acid fall by 32% while thyrotropin releasing hormone levels seem to increase 35% and substance P/substance K levels also increase 48 and 72% respectively. Imipramine treatment resulted in similar although less pronounced changes.(ABSTRACT TRUNCATED AT 250 WORDS)