The tau of MARK: a polarized view of the cytoskeleton.

Microtubule-affinity regulating kinases (MARKs) were originally discovered by their ability to phosphorylate tau protein and related microtubule-associated proteins (MAPs), and thereby to regulate microtubule dynamics in neurons. Members of the MARK (also known as partition-defective [Par]-1 kinase) family were subsequently found to be highly conserved and to have key roles in cell processes such as determination of polarity, cell-cycle control, intracellular signal transduction, transport and cytoskeleton. This is important for neuronal differentiation, but is also prominent in neurodegenerative 'tauopathies' such as Alzheimer's disease. The identified functions of MARK/Par-1 are diverse and require accurate regulation. Recent discoveries including the x-ray structure of human MARKs contributed to an increased understanding of the mechanisms that control the kinase activity and, thus, the actin and microtubule cytoskeleton.

Microtubule affinity-regulating kinase 2 (MARK2) turns on phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) at Thr-313, a mutation site in Parkinson disease: effects on mitochondrial transport.

The kinase MARK2/Par-1 plays key roles in several cell processes, including neurodegeneration such as Alzheimer disease by phosphorylating tau and detaching it from microtubules. In search of interaction partners of MARK2, we identified phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), which is important for the survival of neurons and whose mutations are linked to familial Parkinson disease (PD). MARK2 phosphorylated and activated the cleaved form of PINK1 (ΔN-PINK1; amino acids 156-581). Thr-313 was the primary phosphorylation site, a residue mutated to a non-phosphorylatable form (T313M) in a frequent variant of PD. Mutation of Thr-313 to Met or Glu in PINK1 showed toxic effects with abnormal mitochondrial distribution in neurons. MARK2 and PINK1 were found to colocalize with mitochondria and regulate their transport. ΔN-PINK1 promoted anterograde transport and increased the fraction of stationary mitochondria, whereas full-length PINK1 promoted retrograde transport. In both cases, MARK2 enhanced the effects. The results identify MARK2 as an upstream regulator of PINK1 and ΔN-PINK1 and provide insights into the regulation of mitochondrial trafficking in neurons and neurodegeneration in PD.

Interactions of MAP/microtubule affinity regulating kinases with the adaptor complex AP-2 of clathrin-coated vesicles.

MARK [microtubule-associated proteins (MAPs)/microtubule affinity regulating kinase]/Par-1 (partition defective) phosphorylate MAPs tau, MAP2 and MAP4 at KXGS motifs and thereby regulate microtubule dynamics and transport processes in cells [Drewes et al., Cell1997;89:297-308]. We report here that MARK copurifies with clathrin-coated vesicles (CCVs) via interaction with the adaptor complex AP-2. The adaptin binding site on MARK includes the regulatory loop of its catalytic domain. Immunofluorescence demonstrates the colocalization of MARK with AP-2 and clathrin, as well as other MARK-interacting proteins such as PAK5. The results are consistent with an observed influence of MARK on the trafficking of CCVs. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

PAK5 kinase is an inhibitor of MARK/Par-1, which leads to stable microtubules and dynamic actin.

MARK/Par-1 is a kinase involved in development of embryonic polarity. In neurons, MARK phosphorylates tau protein and causes its detachment from microtubules, the tracks of axonal transport. Because the target sites of MARK on tau occur at an early stage of Alzheimer neurodegeneration, we searched for interaction partners of MARK. Here we report that MARK2 is negatively regulated by PAK5, a neuronal member of the p21-activated kinase family. PAK5 suppresses the activity of MARK2 toward its target, tau protein. The inhibition requires the binding between the PAK5 and MARK2 catalytic domains, but does not require phosphorylation. In transfected Chinese hamster ovary (CHO) cells both kinases show a vesicular distribution with partial colocalization on endosomes containing AP-1/2. Although MARK2 transfected alone destabilizes microtubules and stabilizes actin stress fibers, PAK5 keeps microtubules stable through the down-regulation of MARK2 but destabilizes the F-actin network so that stress fibers and focal adhesions disappear and cells develop filopodia. The results point to an inverse relationship between actin- and microtubule-related signaling by the PAK5 and MARK2 pathways that affect both cytoskeletal networks.

Age-dependent neuroinflammation and cognitive decline in a novel Ala152Thr-Tau transgenic mouse model of PSP and AD.

INTRODUCTION: Mutations of Tau are associated with several neurodegenerative disorders. Recently, the Tau mutation A152T was described as a novel risk factor for frontotemporal dementia spectrum disorders and Alzheimer disease. In vitro Tau-A152T shows a decreased binding to microtubules and a reduced tendency to form abnormal fibers. RESULTS: To study the effects of this mutation we generated a mouse model expressing human full-length Tau with this mutation (hTau40(AT)). At young age (2-3 months) immunohistological analysis reveals pathological Tau conformation and Tau-hyperphosphorylation combined with Tau missorting into the somatodendritic compartment of neurons. With increasing age there is Tau aggregation including co-aggregates of endogenous mouse Tau and exogenous human Tau, accompanied by loss of synapses (especially presynaptic failure) and neurons. From ~10 months onwards the mice show a prominent neuroinflammatory response as judged by activation of microglia and astrocytes. This progressive neuroinflammation becomes visible by in vivo bioluminescence imaging after crossbreeding of hTau40(AT) mice and Gfap-luciferase reporter mice. In contrast to other Tau-transgenic models and Alzheimer disease patients with reduced protein clearance, hTau40(AT) mice show a strong induction of autophagy. Although Tau-hyperphosphorylation and aggregation is also present in spinal cord and motor cortex (due to the Thy1.2 promoter), neuromotor performance is not affected. Deficits in spatial reference memory are manifest at ~16 months and are accompanied by neuronal death. CONCLUSIONS: The hTau40(AT) mice mimic pathological hallmarks of tauopathies including a cognitive phenotype combined with pronounced neuroinflammation visible by bioluminescence. Thus the mice are suitable for mechanistic studies of Tau induced toxicity and in vivo validation of neuroprotective compounds.

Preventive methylene blue treatment preserves cognition in mice expressing full-length pro-aggregant human Tau.

INTRODUCTION: Neurofibrillary tangles (NFT) composed of Tau are hallmarks of neurodegeneration in Alzheimer disease. Transgenic mice expressing full-length pro-aggregant human Tau (2N4R Tau-ΔK280, termed Tau(ΔK)) or its repeat domain (TauRD-ΔK280, TauRD(ΔK)) develop a progressive Tau pathology with missorting, phosphorylation, aggregation of Tau, loss of synapses and functional deficits. Whereas TauRD(ΔK) assembles into NFT concomitant with neuronal death, Tau(ΔK) accumulates into Tau pretangles without overt neuronal loss. Both forms cause a comparable cognitive decline (with onset at 10mo and 12mo, respectively), which is rescued upon switch-off of transgene expression. Since methylene blue (MB) is able to inhibit Tau aggregation in vitro, we investigated whether MB can prevent or rescue Tau-induced cognitive impairments in our mouse models. Both types of mice received MB orally using different preventive and therapeutic treatment protocols, initiated either before or after disease onset. The cognitive status of the mice was assessed by behavior tasks (open field, Morris water maze) to determine the most successful conditions for therapeutic intervention. RESULTS: Preventive and therapeutic MB application failed to avert or recover learning and memory deficits of TauRD(ΔK) mice. Similarly, therapeutic MB treatment initiated after onset of cognitive impairments was ineffective in Tau(ΔK) mice. In contrast, preventive MB application starting before onset of functional deficits preserved cognition of Tau(ΔK) mice. Beside improved learning and memory, MB-treated Tau(ΔK) mice showed a strong decrease of insoluble Tau, a reduction of conformationally changed (MC1) and phosphorylated Tau species (AT180, PHF1) as well as an upregulation of protein degradation systems (autophagy and proteasome). This argues for additional pleiotropic effects of MB beyond its properties as Tau aggregation inhibitor. CONCLUSIONS: Our data support the use of Tau aggregation inhibitors as potential drugs for the treatment of AD and other tauopathies and highlights the need for preventive treatment before onset of cognitive impairments.

Emerging modes of PINK1 signaling: another task for MARK2.

PTEN-induced kinase 1 (PINK1) acts at multiple levels to promote mitochondrial health, including regulatory influence on ATP-synthesis, protein quality control, apoptosis, mitochondrial transport, and destiny. PINK1 mutations are linked to Parkinson disease (PD) and mostly result in loss of kinase activity. But the molecular events responsible for neuronal death as well as the physiological targets and regulators of PINK1 are still a matter of debate. This review highlights the recent progress evolving the cellular functions of the cytosolic pool of PINK1 in mitochondrial trafficking and neuronal differentiation. Regulation of PINK1 signaling occurs by mitochondrial processing to truncated forms of PINK1, differentially targeted to several subcellular compartments. The first identified activating kinase of PINK1 is MAP/microtubule affinity regulating kinase 2 (MARK2), which phosphorylates T313, a frequent mutation site linked to PD. Kinases of the MARK2 family perform diverse functions in neuronal polarity, transport, migration, and neurodegeneration such as Alzheimer disease (AD). This new protein kinase signaling axis might provide a link between neurodegenerative processes in AD and PD diseases and opens novel possibilities in targeting pathological signaling processes.

Spred1 and TESK1--two new interaction partners of the kinase MARKK/TAO1 that link the microtubule and actin cytoskeleton.

The signaling from MARKK/TAO1 to the MAP/microtubule affinity-regulating kinase MARK/Par1 to phosphorylated microtubule associated proteins (MAPs) renders microtubules dynamic and plays a role in neurite outgrowth or polarity development. Because hyperphosphorylation of Tau at MARK target sites is a hallmark of Alzheimer neurodegeneration, we searched for upstream regulators by the yeast two-hybrid approach and identified two new interaction partners of MARKK, the regulatory Sprouty-related protein with EVH-1 domain1 (Spred1) and the testis-specific protein kinase (TESK1). Spred1-MARKK binding has no effect on the activity of MARKK; therefore, it does not change microtubule (MT) stability. Spred1-TESK1 binding causes inhibition of TESK1. Because TESK1 can phosphorylate cofilin and thus stabilizes F-actin stress fibers, the inhibition of TESK1 by Spred1 makes F-actin fibers dynamic. A third element in this interaction triangle is that TESK1 binds to and inhibits MARKK. Thus, in Chinese hamster ovary (CHO) cells the elevation of MARKK results in MT disruption (via activation of MARK/Par1 and phosphorylation of MAPs), but this can be blocked by TESK1. Similarly, enhanced TESK1 activity results in increased stress fibers (via phospho-cofilin), but this can be blocked by elevating Spred1. Thus, the three-way interaction between Spred1, MARKK, and TESK1 represents a pathway that links regulation of both the microtubule- and F-actin cytoskeleton.