Microtubule affinity-regulating kinase 4 with an Alzheimer's disease-related mutation promotes tau accumulation and exacerbates neurodegeneration.

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 Ser-262 and Ser-356 plays 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 Ser-262/356-dependent and -independent mechanisms. Using transgenic Drosophila expressing human MARK4 (MARK4wt) or a mutant version of MARK4 (MARK4ΔG316E317D), we found that coexpression 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 Ser-262 and Ser-356 was blocked by alanine substitutions, MARK4wt did not promote tau accumulation or exacerbate neurodegeneration, whereas coexpression 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 affect disease pathogenesis.

Cdk5 increases MARK4 activity and augments pathological tau accumulation and toxicity through tau phosphorylation at Ser262.

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.

Tau phosphorylation at Alzheimer's disease-related Ser356 contributes to tau stabilization when PAR-1/MARK activity is elevated.

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.

S6K/p70S6K1 protects against tau-mediated neurodegeneration by decreasing the level of tau phosphorylated at Ser262 in a Drosophila model of tauopathy.

Abnormal accumulation of the microtubule-associated protein tau is thought to cause neuronal cell death in a group of age-associated neurodegenerative disorders. Tau is phosphorylated at multiple sites in diseased brains, and phosphorylation of tau at Ser262 initiates tau accumulation and toxicity. In this study, we sought to identify novel factors that affect the metabolism and toxicity of tau phosphorylated at Ser262 (pSer262-tau). A biased screen using a Drosophila model of tau toxicity revealed that knockdown of S6K, the Drosophila homolog of p70S6K1, increased the level of pSer262-tau and enhanced tau toxicity. S6K can be activated by the insulin signaling, however, unlike knockdown of S6K, knockdown of insulin receptor or insulin receptor substrate nonselectively decreased total tau levels via autophagy. Importantly, activation of S6K significantly suppressed tau-mediated axon degeneration, whereas manipulation of either the insulin signaling pathway or autophagy did not. Our results suggest that activation of S6K may be an effective therapeutic strategy for selectively decreasing the levels of toxic tau species and suppressing neurodegeneration.

Ca2+/calmodulin-dependent protein kinase II promotes neurodegeneration caused by tau phosphorylated at Ser262/356 in a transgenic Drosophila model of tauopathy.

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.