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Abnormal phosphorylation of the microtubule-associated protein tau is observed in many neurodegenerative diseases, including Alzheimer’s disease (AD). AD-related phosphorylation of two tau residues, Ser262 and Ser356, by PAR-1/MARK stabilizes tau in the initial phase of mismetabolism, leading to subsequent phosphorylation events, accumulation, and toxicity. However, the relative contribution of phosphorylation at each of these sites to tau stabilization has not yet been elucidated. In a Drosophila model of human tau toxicity, we found that tau was phosphorylated at Ser262, but not at Ser356, and that blocking Ser262 phosphorylation decreased total tau levels. By contrast, when PAR-1 was co-overexpressed with tau, tau was hyperphosphorylated at both Ser262 and Ser356. Under these conditions, the protein levels of tau were significantly elevated, and prevention of tau phosphorylation at both residues was necessary to completely suppress this elevation. These results suggest that tau phosphorylation at Ser262 plays the predominant role in tau stabilization when PAR-1/MARK activity is normal, whereas Ser356 phosphorylation begins to contribute to this process when PAR-1/MARK activity is abnormally elevated, as in diseased brains.

Abnormal deposition of the microtubule-associated protein tau is a common pathological feature of multiple neurodegenerative diseases, including Alzheimer's disease (AD), and plays critical roles in their pathogenesis. Disruption of calcium homeostasis and the downstream kinase Ca2+/calmodulin-dependent protein kinase II (CaMKII) coincides with pathological phosphorylation of tau in AD brains. However, it remains unclear whether and how dysregulation of CaMKII affects tau toxicity. Using a Drosophila model, we found that CaMKII promotes neurodegeneration caused by tau phosphorylated at the AD-associated sites Ser262/356. Overexpression of CaMKII promoted, while RNA-mediated knockdown of CaMKII and inhibition of CaMKII activity by expression of an inhibitory peptide suppressed, tau-mediated neurodegeneration. Blocking tau phosphorylation at Ser262/356 by alanine substitutions suppressed promotion of tau toxicity by CaMKII, suggesting that tau phosphorylation at these sites is required for this phenomenon. However, neither knockdown nor overexpression of CaMKII affected tau phosphorylation levels at Ser262/356, suggesting that CaMKII is not directly involved in tau phosphorylation at Ser262/356 in this model. These results suggest that a pathological cascade of events, including elevated levels of tau phosphorylated at Ser262/356 and aberrant activation of CaMKII, work in concert to promote tau-mediated neurodegeneration.

Brain neurons play a central role in organismal aging, but there is conflicting evidence about the role of neuronal glucose availability because glucose uptake and metabolism are associated with both aging and extended life span. Here, we analyzed metabolic changes in the brain neurons of Drosophila during aging. Using a genetically encoded fluorescent adenosine triphosphate (ATP) biosensor, we found decreased ATP concentration in the neuronal somata of aged flies, correlated with decreased glucose content, expression of glucose transporter and glycolytic enzymes and mitochondrial quality. The age-associated reduction in ATP concentration did not occur in brain neurons with suppressed glycolysis or enhanced glucose uptake, suggesting these pathways contribute to ATP reductions. Despite age-associated mitochondrial damage, increasing glucose uptake maintained ATP levels, suppressed locomotor deficits, and extended the life span. Increasing neuronal glucose uptake during dietary restriction resulted in the longest life spans, suggesting an additive effect of enhancing glucose availability during a bioenergetic challenge on aging.

Accumulation of microtubule-associated tau protein is thought to cause neuron loss in a group of neurodegenerative diseases called tauopathies. In diseased brains, tau molecules adopt pathological structures that propagate into insoluble forms with disease-specific patterns. Several types of posttranslational modifications in tau are known to modulate its aggregation propensity in vitro, but their influence on tau accumulation and toxicity at the whole-organism level has not been fully elucidated. Herein, we utilized a series of transgenic Drosophila models to compare systematically the toxicity induced by five tau constructs with mutations or deletions associated with aggregation, including substitutions at seven disease-associated phosphorylation sites (S7A and S7E), deletions of PHF6 and PHF6* sequences (ΔPHF6 and ΔPHF6*), and substitutions of cysteine residues in the microtubule binding repeats (C291/322A). We found that substitutions and deletions resulted in different patterns of neurodegeneration and accumulation, with C291/322A having a dramatic effect on both tau accumulation and neurodegeneration. These cysteines formed disulfide bonds in mouse primary cultured neurons and in the fly retina, and stabilized tau proteins. Additionally, they contributed to tau accumulation under oxidative stress. We also found that each of these cysteine residues contributes to the microtubule polymerization rate and microtubule levels at equilibrium, but none of them affected tau binding to polymerized microtubules. Since tau proteins expressed in the Drosophila retina are mostly present in the early stages of tau filaments self-assembly, our results suggest that disulfide bond formation by these cysteine residues could be attractive therapeutic targets.

HighlightsIn vivo imaging of Drosophila brain reveals aged neurons suffer from energy deficits.Increased neuronal glucose uptake is sufficient to ameliorate age-dependent declines in ATP. Beneficial effects of increased glucose uptake surpass age-dependent mitochondrial damage.Increased neuronal glucose uptake suppresses age-related locomotor decline and extends lifespan under dietary restriction. SummaryBrain neurons play a central role in organismal aging, but underlying mechanisms are still unclear. Dietary restriction extends lifespan, suggesting that bioenergetic challenges may be beneficial. By contrast, cerebral blood flow and glycolysis decrease during aging, suggesting that brain neurons in aged animals experience an energy deficit.In this study, we analyzed age-associated changes in metabolic states in the brain neurons of Drosophila and how such changes affect aging. Using a genetically encoded fluorescent ATP biosensor, we show that ATP concentration decreases in the neuronal somata of the brain during aging. A decrease in glucose content, expression of glucose transporter and glycolytic enzymes, and mitochondrial quality correlated with the reduction in ATP concentration. The age-associated reduction in ATP concentration did not occur in brain neurons in which glycolysis was suppressed or glucose transporter was overexpressed to enhance glucose uptake, suggesting that lower glucose uptake and glycolysis underlies the reduction in ATP in aged neurons. In contrast, increasing the glucose uptake did not rescue age-associated mitochondrial damage and surpassed mitochondrial defects to maintain ATP levels. Furthermore, increasing neuronal glucose uptake suppressed age-dependent locomotor deficits and extended the life span, and life span extension was more prominent on the low-calorie diet. These results suggest that strategies aimed at increasing the neuronal uptake of glucose may further promote the beneficial effects of bioenergetic challenges on aging.

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