Fish growth rates and lake sulphate explain variation in mercury levels in ninespine stickleback (Pungitius pungitius) on the Arctic Coastal Plain of Alaska.

Mercury concentrations in freshwater food webs are governed by complex biogeochemical and ecological interactions that spatially vary and are often mediated by climate. The Arctic Coastal Plain of Alaska (ACP) is a heterogeneous, lake-rich landscape where variability in mercury accumulation is poorly understood. Earlier research indicated that the level of catchment influence on lakes varied spatially on the ACP, and affected mercury accumulation in lake sediments. This work sought to determine drivers of spatial variation in mercury accumulation in lake food webs on the ACP. Three lakes that were a priori identified as "high catchment influence" (Reindeer Camp region) and three lakes that were a priori identified as "low catchment influence" (Atqasuk region) were sampled, and variability in water chemistry, food web ecology, and mercury accumulation was investigated. Among-lake differences in ninespine stickleback (Pungitius pungitius) length-adjusted methylmercury concentrations were significantly explained by sulphate concentration in lake water, a tracer of catchment runoff input. This effect was mediated by fish growth, which had no pattern between regions. Together, lake water sulphate concentration and fish age-at-size (proxy for growth) accounted for nearly all of the among-lake variability in length-adjusted methylmercury concentrations in stickleback (R2adj = 0.94, p < 0.01). The percentage of total mercury as methylmercury (a proxy for net Hg methylation) was higher in sediments of more autochthonous, "low catchment influence" lakes (p < 0.05), and in the periphyton of more allochthonous, "high catchment influence" lakes (p < 0.05). The results indicate that dominant sources of primary production (littoral macrophyte/biofilm vs. pelagic phytoplankton) and food web structure (detrital vs. grazing) are regulated by catchment characteristics on the ACP, and that this ultimately influences the amount of methylmercury in the aquatic food web. These results have important implications for predicting future mercury concentrations in fish in lakes where fish growth rates and catchment inputs may change in response to a changing climate.

Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells.

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction.

Alpha-Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons.

Many neuropathological and experimental studies suggest that the degeneration of dopaminergic terminals and axons precedes the demise of dopaminergic neurons in the substantia nigra, which finally results in the clinical symptoms of Parkinson disease (PD). The mechanisms underlying this early axonal degeneration are, however, still poorly understood. Here, we examined the effects of overexpression of human wildtype alpha-synuclein (αSyn-WT), a protein associated with PD, and its mutant variants αSyn-A30P and -A53T on neurite morphology and functional parameters in rat primary midbrain neurons (PMN). Moreover, axonal degeneration after overexpression of αSyn-WT and -A30P was analyzed by live imaging in the rat optic nerve in vivo. We found that overexpression of αSyn-WT and of its mutants A30P and A53T impaired neurite outgrowth of PMN and affected neurite branching assessed by Sholl analysis in a variant-dependent manner. Surprisingly, the number of primary neurites per neuron was increased in neurons transfected with αSyn. Axonal vesicle transport was examined by live imaging of PMN co-transfected with EGFP-labeled synaptophysin. Overexpression of all αSyn variants significantly decreased the number of motile vesicles and decelerated vesicle transport compared with control. Macroautophagic flux in PMN was enhanced by αSyn-WT and -A53T but not by αSyn-A30P. Correspondingly, colocalization of αSyn and the autophagy marker LC3 was reduced for αSyn-A30P compared with the other αSyn variants. The number of mitochondria colocalizing with LC3 as a marker for mitophagy did not differ among the groups. In the rat optic nerve, both αSyn-WT and -A30P accelerated kinetics of acute axonal degeneration following crush lesion as analyzed by in vivo live imaging. We conclude that αSyn overexpression impairs neurite outgrowth and augments axonal degeneration, whereas axonal vesicle transport and autophagy are severely altered.

ROCK2 is a major regulator of axonal degeneration, neuronal death and axonal regeneration in the CNS.

The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increased survival of RGCs after optic nerve axotomy. Moreover, knockdown of ROCK2 attenuated axonal degeneration of the proximal axon after ONC assessed by in vivo live imaging. Mechanistically, we demonstrate here that knockdown of ROCK2 resulted in decreased intraneuronal activity of calpain and caspase 3, whereas levels of pAkt and collapsin response mediator protein 2 and autophagic flux were increased. Taken together, our data characterize ROCK2 as a specific therapeutic target in neurodegenerative diseases and demonstrate new downstream effects of ROCK2 including axonal degeneration, apoptosis and autophagy.

An in vitro model of ethanol-dependent liver cell injury.

Primary cultures of adult rat hepatocytes were incubated (6 to 96 hr) with 50 to 150 mmol/L ethanol, 0.5 mmol/L linoleate, 0.5 mmol/L palmitate, 0.5 mmol/L 4-methylpyrazole, 0 to 25 mumol/L vitamin E phosphate or selected combinations of these agents. Agent-dependent changes in liver cell viability (AST release and reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and function (phospholipid peroxidation, hydrolysis, biosynthesis and triacylglycerol biosynthesis) were determined. The influence of ethanol on liver cell function and viability was dose and incubation time dependent. Short periods (24 hr or less) of exposure to 100 mmol/L ethanol increased liver cell triacylglycerol biosynthesis and phospholipid hydrolysis, peroxidation and biosynthesis without altering cell viability. However, longer periods (72 hr or more) of exposure to 100 or 150 mmol/L ethanol resulted in significant reductions (30% to 50%) in cell viability, function and phosphatidylcholine biosynthesis and content. The ethanol-dependent decreases in cell function and viability were potentiated by linoleate and reduced by vitamin E phosphate, palmitate and 4-methylpyrazole. These results suggest that ethanol-induced liver cell injury in vitro is not a result of ethanol per se, but factors such as acetaldehyde or oxyradicals produced as a consequence of ethanol metabolism. Therefore the incubation of cultured hepatocytes with ethanol may be an appropriate model in vitro for determining the mechanisms by which ethanol intake disrupts liver cell function in vivo.

An enzymatic explanation of the differential effects of oleate and gemfibrozil on cultured hepatocyte triacylglycerol and phosphatidylcholine biosynthesis and secretion.

Incubation (1-4 h) of primary cultures of adult rat hepatocytes with gemfibrozil (0.1-1.0 mM) significantly decreased the: (1) incorporation of [1,3-14C]glycerol into cellular triacylglycerol (30%); (2) secretion of labeled (VLDL) triacylglycerol (4-fold); and (3) oleate-induced rise in triacylglycerol biosynthesis and secretion. Gemfibrozil also increased the: (1) incorporation of labeled glycerol into cellular phosphatidylcholine (2-fold); and (2) secretion of labeled (HDL) phosphatidylcholine (10-fold). The gemfibrozil-dependent increase in the flux of labeled diacylglycerol into phosphatidylcholine is rapid (15 min) and associated with a 2-fold increase in membrane-bound phosphocholine cytidylyltransferase activity. A phosphocholine cytidylyltransferase-mediated rise in cellular CDP choline content may explain the gemfibrozil-dependent rise in phosphatidylcholine biosynthesis since homogenates of monolayers incubated with CDP choline preferentially incorporate labeled diacylglycerol into phosphatidylcholine rather than triacylglycerol. Therefore, the triacylglycerol-lowering potential of gemfibrozil may be due in part to its ability to shunt liver cell diacylglycerol into phosphatidylcholine rather than triacylglycerol. These results suggest that CDP choline may be a key regulator of the diacylglycerol branchpoint, since diacylglycerol is primarily incorporated into phosphatidylcholine or triacylglycerol depending on whether CDP choline is or is not available.