Accumulation of the microtubule-associated protein tau is associated with Alzheimer’s disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser262 and Ser356 play critical roles in tau accumulation and toxicity. Microtubule-affinity regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317D, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK4ΔG316E317D increases the abundance of highly phosphorylated, insoluble tau species, and exacerbates neurodegeneration via Ser262/356-dependent and -independent mechanisms. Using transgenic Drosophila expressing human MARK4 (MARK4wt) or a mutant version of MARK4 (MARK4ΔG316E317D), we found that co-expression of MARK4wt and MARK4ΔG316E317D increased total tau levels and enhanced tau-induced neurodegeneration, and that MARK4ΔG316E317D had more potent effects than MARK4wt. Interestingly, the in vitro kinase activities of MARK4wt and MARK4ΔG316E317D were similar. When tau phosphorylation at Ser262 and Ser356 was blocked by alanine substitutions, MARK4wt did not promote tau accumulation or exacerbate neurodegeneration, while co-expression of MARK4ΔG316E317D did. Both MARK4wt and MARK4ΔG316E317D increased the levels of oligomeric forms of tau; however, only MARK4ΔG316E317D further increased the detergent insolubility of tau in vivo. Together, these findings suggest that MARK4ΔG316E317D increases tau levels and exacerbates tau toxicity via a novel gain-of-function mechanism, and that modification in this region of MARK4 may impact disease pathogenesis.
Hyperphosphorylation of the microtubule-associated protein tau is associated with many neurodegenerative diseases, including Alzheimer’s disease. Microtubule affinity-regulating kinases (MARK) 1–4 and cyclin-dependent kinase 5 (Cdk5) are tau kinases under physiological and pathological conditions. However, their functional relationship remains elusive. Here, we report a novel mechanism by which Cdk5 activates MARK4 and augments tau phosphorylation, accumulation and toxicity. MARK4 is highly phosphorylated at multiple sites in the brain and in cultured neurons, and inhibition of Cdk5 activity reduces phosphorylation levels of MARK4. MARK4 is known to be activated by phosphorylation at its activation loop by liver kinase B1 (LKB1). In contrast, Cdk5 increased phosphorylation of MARK4 in the spacer domain, but not in the activation loop, and enhanced its kinase activity, suggesting a novel mechanism by which Cdk5 regulates MARK4 activity. We also demonstrated that co-expression of Cdk5 and MARK4 in mammalian cultured cells significantly increased the levels of tau phosphorylation at both Cdk5 target sites (SP/TP sites) and MARK target sites (Ser262), as well as the levels of total tau. Furthermore, using a Drosophila model of tau toxicity, we demonstrated that Cdk5 promoted tau accumulation and tau-induced neurodegeneration via increasing tau phosphorylation levels at Ser262 by a fly ortholog of MARK, Par-1. This study suggests a novel mechanism by which Cdk5 and MARK4 synergistically increase tau phosphorylation and accumulation, consequently promoting neurodegeneration in disease pathogenesis.
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.
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