Effects of oxidized and reduced forms of methylthioninium in two transgenic mouse tauopathy models

Acknowledgements The authors acknowledge the contributions of Bettina Seelhorst (histological analysis), Anna Thoma (animal care), Marlene Arthur (animal dosing) and Pierre-Henri Moreau (experimental discussions). This work was supported by TauRx Therapeutics Ltd., Singapore.Peer reviewedPublisher PD

Functional genomic screen and network analysis reveal novel modifiers of tauopathy dissociated from tau phosphorylation

A functional genetic screen using loss-of-function and gain-of-function alleles was performed to identify modifiers of tau-induced neurotoxicity using the 2N/4R (full-length) isoform of wild-type human tau expressed in the fly retina. We previously reported eye pigment mutations, which create dysfunctional lysosomes, as potent modifiers; here, we report 37 additional genes identified from ∼1900 genes screened, including the kinases shaggy/GSK-3beta, par-1/MARK, CamKI and Mekk1. Tau acts synergistically with Mekk1 and p38 to down-regulate extracellular regulated kinase activity, with a corresponding decrease in AT8 immunoreactivity (pS202/T205), suggesting that tau can participate in signaling pathways to regulate its own kinases. Modifiers showed poor correlation with tau phosphorylation (using the AT8, 12E8 and AT270 epitopes); moreover, tested suppressors of wild-type tau were equally effective in suppressing toxicity of a phosphorylation-resistant S11A tau construct, demonstrating that changes in tau phosphorylation state are not required to suppress or enhance its toxicity. Genes related to autophagy, the cell cycle, RNA-associated proteins and chromatin-binding proteins constitute a large percentage of identified modifiers. Other functional categories identified include mitochondrial proteins, lipid trafficking, Golgi proteins, kinesins and dynein and the Hsp70/Hsp90-organizing protein (Hop). Network analysis uncovered several other genes highly associated with the functional modifiers, including genes related to the PI3K, Notch, BMP/TGF-β and Hedgehog pathways, and nuclear trafficking. Activity of GSK-3β is strongly upregulated due to TDP-43 expression, and reduced GSK-3β dosage is also a common suppressor of Aβ42 and TDP-43 toxicity. These findings suggest therapeutic targets other than mitigation of tau phosphorylation

Expression of aquaporin-1 in human trabecular meshwork cells: role in resting cell volume. Invest Ophthalmol Vis Sci 42: 1803–1811

PURPOSE. Drainage of aqueous humor from the human eye appears dependent on intracellular volume of trabecular meshwork (TM) cells, the predominant cell type of the human outflow pathway. Thus, the modulation of water and solute flux across the plasma membrane of TM cells is predicted to be an important factor in regulating outflow facility. Aquaporin (AQP)-1 is a hexahelical integral membrane protein that functions as a regulated channel for water and cations in fluidsecreting and -absorbing tissues. AQP1 is present in many tissues of the human eye, including the TM; however, its role in outflow facility is unknown. The purpose of the present study was twofold: to evaluate the prospect of manipulating AQP1 protein levels in TM cells using sense and antisense mRNA and to investigate the functional role of AQP1 in TM cells. METHODS. An adenovirus (AV) expression system was used to alter AQP1 protein levels. AQP1 protein expression was monitored using immunoblot analysis, and resting cell volume was measured by forward light scatter, electronic cell sizing, and [ 14 C]-sucrose/urea equilibration. Permeability of TM monolayers to [ 14 C]-sucrose was also assessed as an indirect evaluation of cell volume. RESULTS. AV-mediated gene transfer of AQP1 cDNA to TM cells resulted in a titer-dependent increase in recombinant AQP1, whereas transfer of antisense cDNA decreased native AQP1 protein by 71.7% Ϯ 5.5% (P Ͻ 0.01) after 5 days. A novel finding of this study is that mean resting volumes of AQP1(s) AV-infected TM cells in suspension were 8.7% Ϯ 3.0% greater (P Ͻ 0.05) than control cells. Conversely, AQP1 antisense (as) AV-infected cells had resting volumes 7.8% Ϯ 2.9% less than control cells (P Ͻ 0.05). Similar effects of AQP1 expression on resting cell volume were observed in TM monolayers. Consistent with this finding, paracellular permeability of AQP1(s) AV-infected TM monolayers to [ 14 C]-sucrose decreased by 8.0% Ϯ 1.4% (P Ͻ 0.001). CONCLUSIONS. In addition to influencing the osmotic permeability of TM plasma membranes, the level of AQP1 protein expression influences resting intracellular volume and thus paracellular permeability of TM cell monolayers in vitro. These data suggest that AQP1 expression may affect outflow facility in vivo. (Invest Ophthalmol Vis Sci. 2001;42:1803-1811 A quaporin (AQP)-1 is a hexahelical integral membrane protein that functions as a regulated channel for water and cations. 9 In the human eye, AQP1 is present in many tissues that require the efficient movement of water, including the corneal endothelium, the lens epithelium, the iris epithelium, the nonpigmented ciliary epithelium, and the cells of the conventional outflow pathway. 9,10 The conventional outflow pathway contains two cell types, trabecular meshwork (TM) and Schlemm's canal (SC) endothelial cells, both of which express AQP1. -11 TM cells cover collagen lamellae that form the maze of passages through which water must flow in the outflow pathway. As aqueous humor drains from the eye, it first passes through the tortuous TM until finally reaching and crossing a monolayer of endothelial cells that line SC. Because aqueous humor appears to move primarily by bulk flow around TM cells and not through them (i.e., paracellular rather than transcellular), 13-15 For example, agents that decrease TM-cell volume increase outflow facility, and agents that increase TM-cell volume decrease outflow facility. These findings suggest that intracellular volume of TM cells in vivo may dictate outflow resistance by affecting the dimensions or direction of the human outflow pathway; particularly in the juxtacanalicular region, where spaces between TM cells approach 1 m. Whether AQP1 plays a physiological role in outflow across TM cells is unknown. Defining the functional contribution of AQP1 to the permeability of TM tissue and to outflow facility may be important for understanding the pathologic course of glaucoma, the second leading cause of blindness in the United States