Functional Interactions of Tau Phosphorylation Sites That Mediate Toxicity and Deficient Learning in Drosophila melanogaster

Abstract

Hyperphosphorylated Tau protein is the main component of the neurofibrillary tangles, characterizing degenerating neurons in Alzheimer's disease and other Tauopathies. Expression of human Tau protein in Drosophila CNS results in increased toxicity, premature mortality and learning and memory deficits. Herein we use novel transgenic lines to investigate the contribution of specific phosphorylation sites previously implicated in Tau toxicity. These three different sites, Ser 238 , Thr 245 , and Ser 262 were tested either by blocking their phosphorylation, by Ser/Thr to Ala substitution, or pseudophosphorylation, by changing Ser/Thr to Glu. We validate the hypothesis that phosphorylation at Ser 262 is necessary for Tau-dependent learning deficits and a "facilitatory gatekeeper" to Ser 238 occupation, which is linked to Tau toxicity. Importantly we reveal that phosphorylation at Thr 245 acts as a "suppressive gatekeeper", preventing phosphorylation of many sites including Ser 262 and consequently of Ser 238. Therefore, we elucidate novel interactions among phosphosites central to Tau mediated neuronal dysfunction and toxicity, likely driven by phosphorylation-dependent conformational plasticity.

The Two Cysteines of Tau Protein Are Functionally Distinct and Contribute Differentially to Its Pathogenicityin Vivo

“…6.0886, p = 0.0062; subsequent LSM: p = 0.0015 and p = 0.1429 vs controls, respectively; and C322A ANOVA: F (2,39) = 14.5887, p , 0.0001; subsequent LSM: p = 0.2914 and p = 7.52e-06 vs controls, respectively). These results indicate that blocking cysteines of Tau confers a crucial role for this protein in neuronal dysfunction and support the proposal that elevated occupation of phosphor-Ser262 is indeed requisite for the learning deficits (Papanikolopoulou and Skoulakis, 2015;Keramidis et al, 2020).…”

“…Drosophila culture and strains Flies were cultured in standard sugar-wheat flour food supplemented with soy flour and CaCl 2 (Acevedo et al, 2007). Pan-neuronal transgene expression was achieved using the Elav C155 -Gal4;Ras2-Gal4 driver (Robinow and White, 1988;Walker et al, 2006;Keramidis et al, 2020). The double driver was constructed using standard genetic crosses.…”

“…Neuronal homeostatic responses to environmental stressors are essential for maintaining robust behavioral outputs, and Tau-induced toxicity has been shown to be accompanied by impaired neuronal fitness and function (Papanikolopoulou and Skoulakis, 2011). Flies experiencing increased oxidative stress because of pathologic hTau accumulation have increased susceptibility to oxidative injury (Dias-Santagata et al, 2007;Keramidis et al, 2020). To that end, we first tested whether Tau mutant transgenic flies manifest altered responses to oxidative stress.…”

“…Both cysteine residues impact hTau-associated dysfunction Insights into Tau-mediated neuronal dysfunction can be assayed in the Drosophila model systems through investigation of learning and memory (Kosmidis et al, 2010;Papanikolopoulou and Skoulakis, 2015;Sealey et al, 2017). Interestingly, phosphorylation at Ser-262 was proposed to be necessary for and predict learning deficits (Papanikolopoulou and Skoulakis, 2015;Keramidis et al, 2020); and given that this site is hypophosphorylated in the two mutants, we hypothesized that cysteine substitution could impact the cognitive performance of animals. To examine associative learning and memory, we used the olfactory classical conditioning paradigm (Tully and Quinn, 1985) and tested control and Tau-expressing animals immediately after training to assess 3 min memory and 24 h later to probe consolidated memories.…”

Mical modulates Tau toxicity via cysteine oxidation in vivo

“…Flies were cultured in standard sugar-wheat flour food supplemented with soy flour and CaCl 2 [ 26 ]. Panneuronal transgene expression was achieved using the elav C155 -GAL4 or the elav C155 -GAL4;Ras2-GAL4 double driver as already described [ 7 , 27 ]. Fly crosses and experiments were performed at 25 °C unless noted otherwise.…”