Tau Tubulin Kinase Research Articles

Tau tubulin kinase is required for spermatogenesis and development of motile cilia in planarian flatworms.

Cilia are microtubule-based structures that protrude from the apical surface of cells to mediate motility, transport, intracellular signaling, and environmental sensing. Tau tubulin kinases (TTBKs) destabilize microtubules by phosphorylating microtubule-associated proteins (MAPs) of the MAP2/Tau family, but also contribute to the assembly of primary cilia during embryogenesis. Expression of TTBKs is enriched in testicular tissue, but their relevance to reproductive processes is unknown. We identified six TTBK homologues in the genome of the planarian Schmidtea mediterranea (Smed-TTBK-a, -b, -c, -d, -e, and -f), all of which are preferentially expressed in testes. Inhibition of TTBK paralogues by RNA interference (RNAi) revealed a specific requirement for Smed-TTBK-d in postmeiotic regulation of spermatogenesis. Disrupting expression of Smed-TTBK-d results in loss of spermatozoa, but not spermatids. In the soma, Smed-TTBK-d RNAi impaired the function of multiciliated epidermal cells in propelling planarian movement, as well as the osmoregulatory function of protonephridia. Decreased density and structural defects of motile cilia were observed in the epidermis of Smed-TTBK-d(RNAi) by phase contrast, immunofluorescence, and transmission electron microscopy. Altogether, these results demonstrate that members of the TTBK family of proteins are postmeiotic regulators of sperm development and also contribute to the formation of motile cilia in the soma.

Tau tubulin kinases in proteinopathy.

A number of neurodegenerative diseases are characterized by deposition of abnormally phosphorylated tau or TDP-43 in disease-affected neurons. These diseases include Alzheimer's disease, frontotemporal lobar degeneration, and amyotrophic lateral sclerosis. No disease-modifying therapeutics is available to treat these disorders, and we have a limited understanding of the cellular and molecular factors integral to disease initiation or progression. Phosphorylated tau and TDP-43 are important markers of pathology in dementia disorders and directly contribute to tau- and TDP-43-related neurotoxicity and neurodegeneration. Here, we review the scope of tau and TDP-43 phosphorylation in neurodegenerative disease and discuss recent work demonstrating the kinases TTBK1 and TTBK2 phosphorylate both tau and TDP-43, promoting neurodegeneration.

Identification of human tau-tubulin kinase 1 inhibitors: an integrated e-pharmacophore-based virtual screening and molecular dynamics simulation

Tau-tubulin kinase 1 inhibitors inhibit tau protein phosphorylation on Ser198, Ser199, Ser202, Ser422, and also in paired helical filaments. We developed receptor-based pharmacophore models by exploiting three TTBK1 protein structures, i.e., 4NFN, 4BTM, and 4BTK. The integrated e-pharmacophore based virtual screening and molecular dynamics simulation recognized four hits viz. ZINC14644839, ZINC00012956, ZINC91332506, and ZINC69775110 as TTBK1 inhibitors. The Glide XP docking energies (−8.48 to −10.71 kcal.mol−1) of hits were better than cocrystal ligand of 4NFN protein structure (−8.37 kcal.mol−1). Among the hits, ZINC14644839 possessed best binding energy with four hydrogen bonding interactions. The inhibitors showed acceptable calculated ADME and blood-brain barrier permeability properties and could be potential TTBK1 inhibitors for neurodegenerative diseases.Communicated by Ramaswamy H. Sarma

MiR-219-5p inhibits tau phosphorylation by targeting TTBK1 and GSK-3β in Alzheimer's disease.

As the most common neurodegenerative disease, Alzheimer's disease (AD) is characterized by memory, perception, and behavioral damage, which may ultimately lead to emotional fluctuation and even lethal delirium. Increasing studies indicate that microRNAs (miRNAs) are associated with pathological features of AD. However, the role of miR-219-5p in AD progression is still unclear. In this study, the functions of miR-219-5p were analyzed in vitro and in vivo. miR-219-5p was notably overexpressed in brain tissues of patients with AD. The overexpression of miR-219-5p activated the phosphorylation of Tau-Ser198, Tau-Ser199, Tau-Ser201, and Tau-Ser422. We further showed that miR-219-5p could mediate a decrease in the protein levels of tau-tubulin kinase 1 (TTBK1) and glycogen synthase kinase 3β (GSK-3β) by directly binding to their 3'-untranslated region, thereby promoting the phosphorylation of tau in SH-SY5Y Cells. Rescue experiments further revealed that the phosphorylation of tau-mediated by miR-219-5p was dependent on the inhibition of TTBK1 and GSK-3β. Moreover, suppressing the expression of both TTBK1 and GSK-3β using miR-219-5p remarkably rescued AD-like symptoms in amyloid precursor protein/presenilin 1 mice. Our findings indicate that the upregulation of TTBK1 and GSK-3β mediated by the loss of miR-219-5p is a possible mechanism that contributes to tau phosphorylation and AD progression.

Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability.

Spinocerebellar ataxia type 11 (SCA11) is a rare, dominantly inherited human ataxia characterized by atrophy of Purkinje neurons in the cerebellum. SCA11 is caused by mutations in the gene encoding the Serine/Threonine kinase Tau tubulin kinase 2 (TTBK2) that result in premature truncations of the protein. We previously showed that TTBK2 is a key regulator of the assembly of primary cilia in vivo. However, the mechanisms by which the SCA11-associated mutations disrupt TTBK2 function, and whether they interfere with ciliogenesis were unknown. In this work, we present evidence that SCA11-associated mutations are dominant negative alleles and that the resulting truncated protein (TTBK2SCA11) interferes with the function of full length TTBK2 in mediating ciliogenesis. A Ttbk2 allelic series revealed that upon partial reduction of full length TTBK2 function, TTBK2SCA11 can interfere with the activity of the residual wild-type protein to decrease cilia number and interrupt cilia-dependent Sonic hedgehog (SHH) signaling. Our studies have also revealed new functions for TTBK2 after cilia initiation in the control of cilia length, trafficking of a subset of SHH pathway components, including Smoothened (SMO), and cilia stability. These studies provide a molecular foundation to understand the cellular and molecular pathogenesis of human SCA11, and help account for the link between ciliary dysfunction and neurodegenerative diseases.

M-Phase Phosphoprotein 9 regulates ciliogenesis by modulating CP110-CEP97 complex localization at the mother centriole

The primary cilium is elongated from the mother centriole and has diverse signaling roles during development and disease. The CP110-CEP97 complex functions as a negative regulator of ciliogenesis, although the mechanisms regulating its mother centriole localization are poorly understood. Here we show that M-Phase Phosphoprotein 9 (MPP9) is recruited by Kinesin Family Member 24 (KIF24) to the distal end of mother centriole where it forms a ring-like structure and recruits CP110-CEP97 by directly binding CEP97. Loss of MPP9 causes abnormal primary cilia formation in growing cells and mouse kidneys. After phosphorylation by Tau Tubulin Kinase 2 (TTBK2) at the beginning of ciliogenesis, MPP9 is targeted for degradation via the ubiquitin-proteasome system, which facilitates the removal of CP110 and CEP97 from the distal end of the mother centriole. Thus, MPP9 acts as a regulator of ciliogenesis by regulating the localization of CP110-CEP97 at the mother centriole.

Tau Tubulin Kinase 1 (TTBK1), a new player in the fight against neurodegenerative diseases

Tau-tubuline kinases (TTBK) are a family of serine/threonine and tyrosine kinases recently discovered and implicated in the phosphorylation of important substrates such as tau, tubuline or TDP-43. Its two homologs, TTBK1 and TTBK2, show different expression patterns and different involvements in physiological mechanisms of great importance such as mitosis, ciliogenesis and neurotransmission. Their phosphorylation activity has also linked them to the development of neurodegenerative diseases like Alzheimer's disease, amyotrophic lateral sclerosis or spinocerebellar ataxia type 11. There are currently only three inhibitors of these kinases described in the literature. This review intends to give an overview of the structure, expression, physiological and pathological mechanisms of both kinases as well as an extended analysis on the molecules that can inhibit them. The final analysis of all this information led us to propose TTBK1 as a new target for the treatment of neurodegenerative diseases and its selective inhibitors as potential effective drugs for the treatment of these severe unmet disorders.

Pathological phosphorylation of tau and TDP-43 by TTBK1 and TTBK2 drives neurodegeneration

BackgroundProgressive neuron loss in the frontal and temporal lobes of the cerebral cortex typifies frontotemporal lobar degeneration (FTLD). FTLD sub types are classified on the basis of neuronal aggregated protein deposits, typically containing either aberrantly phosphorylated TDP-43 or tau. Our recent work demonstrated that tau tubulin kinases 1 and 2 (TTBK1/2) robustly phosphorylate TDP-43 and co-localize with phosphorylated TDP-43 in human postmortem neurons from FTLD patients. Both TTBK1 and TTBK2 were initially identified as tau kinases and TTBK1 has been shown to phosphorylate tau epitopes commonly observed in Alzheimer’s disease and other tauopathies.MethodsTo further elucidate how TTBK1/2 activity contributes to both TDP-43 and tau phosphorylation in the context of the neurodegeneration seen in FTLD, we examined the consequences of elevated human TTBK1/2 kinase expression in transgenic animal models of disease.ResultsWe show that C. elegans co-expressing tau/TTBK1 tau/TTBK2, or TDP-43/TTBK1 transgenes in combination exhibit synergistic exacerbation of behavioral abnormalities and increased pathological protein phosphorylation. We also show that C. elegans co-expressing tau/TTBK1 or tau/TTBK2 transgenes in combination exhibit aberrant neuronal architecture and neuron loss. Surprisingly, the TTBK2/TDP-43 transgenic combination showed no exacerbation of TDP-43 proteinopathy related phenotypes. Additionally, we observed elevated TTBK1/2 protein expression in cortical and hippocampal neurons of FTLD-tau and FTLD-TDP cases relative to normal controls.ConclusionsOur findings suggest a possible etiology for the two most common FTLD subtypes through a kinase activation driven mechanism of neurodegeneration.

Synapse Innervation and Associative Memory Cell Are Recruited for Integrative Storage of Whisker and Odor Signals in the Barrel Cortex through miRNA-Mediated Processes.

Associative learning is a common way for information acquisition, and the integrative storage of multiple associated signals is essential for associative thinking and logical reasoning. In terms of the cellular mechanism for associative memory, our studies by behavioral task and cellular imaging demonstrate that paired whisker and odor stimulations lead to odorant-induced whisker motion and associative memory cell recruitment in the barrel cortex (BC), which is driven presumably by synapse innervation from co-activated sensory cortices. To confirm these associative memory cells and synapse innervations essential for associative memory and to examine their potential mechanisms, we studied a causal relationship between epigenetic process and memory cell/synapse recruitment by manipulating miRNAs and observing the changes from the recruitments of associative memory cells and synapse innervations to associative memory. Anti-miRNA-324 and anti-miRNA-133a in the BC significantly downregulate new synapse innervation, associative memory cell recruitment and odorant-induced whisker motion, where Tau-tubulin kinase-1 expression is increased. Therefore, the upregulated miRNA-324 in associative learning knocks down Ttbk1-mediated Tau phosphorylation and microtubule depolymerization, which drives the balance between polymerization and depolymerization toward the axon prolongation and spine stabilization to initiate new synapse innervations and to recruit associative memory cells.

The small GTPase RSG1 controls a final step in primary cilia initiation.

Primary cilia, which are essential for normal development and tissue homeostasis, are extensions of the mother centriole, but the mechanisms that remodel the centriole to promote cilia initiation are poorly understood. Here we show that mouse embryos that lack the small guanosine triphosphatase RSG1 die at embryonic day 12.5, with developmental abnormalities characteristic of decreased cilia-dependent Hedgehog signaling. Rsg1 mutant embryos have fewer primary cilia than wild-type embryos, but the cilia that form are of normal length and traffic Hedgehog pathway proteins within the cilium correctly. Rsg1 mother centrioles recruit proteins required for cilia initiation and dock onto ciliary vesicles, but axonemal microtubules fail to elongate normally. RSG1 localizes to the mother centriole in a process that depends on tau tubulin kinase 2 (TTBK2), the CPLANE complex protein Inturned (INTU), and its own GTPase activity. The data suggest a specific role for RSG1 in the final maturation of the mother centriole and ciliary vesicle that allows extension of the ciliary axoneme.

The metal and metalloprotein profile of human plasma as biomarkers for stroke diagnosis

Stroke, a major cause of disability and mortality, affects someone in the United States every 40s. Stroke biomarkers, including those that could be used as a blood test for diagnosis of stroke, have been particularly elusive. We performed a double blind study to identify human plasma biomarkers for the diagnosis of stroke, including acute ischemic stroke (AIS) and intracerebral hemorrhage (ICH). We utilized a three-track approach based on the total metal profile, the metal cofactor levels among metalloproteins, and the identification of stroke-related metalloproteins. The study included 14 case-control pairs of AIS and 23 case-control pairs of ICH. Controls were matched to cases based on gender, ethnicity, and age (±5 years). AIS cases were statistically higher from their respective controls for protein bound co-factors Se and Cd, while unique correlations of metal cofactor concentrations among metalloproteins were identified between Pb-W, Sr-W, Pb-V, and Cu-V. ICH cases were statistically higher from their respective controls for Se and Co cofactors, whereas Cd and Pb were statistically lower. Unique correlations between metal cofactors for ICH cases were identified between Pb-W, Sr-W, Pb-V, and Cu-V. Stroke-related metalloproteins were identified, including calpain-15, protein-activated inward rectifier potassium channel 1, tau-tubulin kinase 1, and voltage-dependent L-type calcium channel subunit beta-3. Linear discriminant analysis (LDA) was able to classify patients between stroke cases or controls with 93% accuracy as well as classify patients with one of the four stroke groups with 85% accuracy. Additionally, this study found utmost importance in vanadium (V) and tungsten (W) correlations for both bound and total metal concentrations, suggestive of binding to transferrin or inhibition of oxidoreductases. Future work in stroke patients will seek to quantify varying selenoproteins, including selenoprotein P and glutathione peroxidase and identified zinc finger tissue leakage proteins, and further explore the role of trace metal fluctuations with transferrin.

Associative Memory Cells are Recruited to Encode Triple Sensory Signals via Synapse Formation

Associative learning is a common form of information acquisition, and associative memory is essential for logical reasoning and associative thinking. In addition to memory cells for two associated signals, here we report the recruitment of associative memory cells that encode triple sensory signals. Paired mouse whisker, olfaction and tail stimulations lead to odorant-induced and tail-induced whisker motions. In the mice of showing cross-modal reflexes, barrel cortical neurons and astrocytes become to encode odor and tail signals alongside whisker signal. Such neurons receive the new synapse innervations from the axons of the piriform and S1-tail cortical neurons, in addition to the innate synapse innervations from the axons of the thalamic neurons. The formation of new synapse innervations and the recruitment of associative memory cells require the upregulations of miRNA-324-5p and miRNA-133a-3p that downregulate tau-tubulin kinase-1 (Ttbk1) and methylcytosine dioxygenase Tet3. The associated activations of the sensory cortices elicit their mutual synapse innervations that recruit memory cells to store triple associated signals via epigenetic processes. Furthermore, after the extinction of cross-modal reflexes, the refinements of the synapses and memory cells in the barrel cortex are well maintained, while plasticity in the M1-motor cortex decreases. These results indicate that the acquired signals remain stored in the sensory cortices and the decay of memory retrieval is due to the decreased refinement of their downstream cortical regions in neural circuits from the sensory cortices to the memory-presentation cortices. In terms of the significance of our discoveries, the storages of multiple signals in individual neurons may expand memory volumes, strengthen cognition capabilities and facilitate creative inspiration productions. Information storages in the sensory cortices confer them to signify the sources of the retrieved memory signals. This study is supported by National Basic Research Program (2013CB531304 and 2016YFC1307100) and Natural Science Foundation China (81671071 and 81471123) to JHW.

TAU TUBULIN KINASE ACTIVATION COINCIDES WITH NEUROPATHOLOGY IN DEMENTIA

TAU TUBULIN KINASE ACTIVATION COINCIDES WITH NEUROPATHOLOGY IN DEMENTIA

A Forward Genetic Screen for Molecules Involved in Pheromone-Induced Dauer Formation in Caenorhabditis elegans.

Animals must constantly assess their surroundings and integrate sensory cues to make appropriate behavioral and developmental decisions. Pheromones produced by conspecific individuals provide critical information regarding environmental conditions. Ascaroside pheromone concentration and composition are instructive in the decision of Caenorhabditis elegans to either develop into a reproductive adult or enter into the stress-resistant alternate dauer developmental stage. Pheromones are sensed by a small set of sensory neurons, and integrated with additional environmental cues, to regulate neuroendocrine signaling and dauer formation. To identify molecules required for pheromone-induced dauer formation, we performed an unbiased forward genetic screen and identified phd (pheromone response-defective dauer) mutants. Here, we describe new roles in dauer formation for previously identified neuronal molecules such as the WD40 domain protein QUI-1 and MACO-1 Macoilin, report new roles for nociceptive neurons in modulating pheromone-induced dauer formation, and identify tau tubulin kinases as new genes involved in dauer formation. Thus, phd mutants define loci required for the detection, transmission, or integration of pheromone signals in the regulation of dauer formation.

Tau Tubulin Kinase TTBK2 Sensitivity of Glutamate Receptor GluK2

Background/Aims: Inherited, autosomal dominant spinocerebellar ataxia type 11 (SCA11) is caused by loss of function mutations of TTBK2 (tau tubulin kinase 2). Mutations observed in patients with SCA11 include truncated TTBK2(450). The present study explored the possibility that TTBK2 influences the function of the glutamate receptor GluK2. Methods: GluK2 was expressed in Xenopus oocytes without and with additional expression of wild type TTBK2, the truncated mutant TTBK2(450), or the kinase dead mutants TTBK2(KD) and TTBK2(450/KD). GluK2 current was determined by dual electrode voltage clamp and GluK2 protein abundance in the cell membrane utilizing confocal microscopy. Results: Glutamate exposure of GluK2 expressing oocytes generated a current, which was significantly lower in oocytes expressing GluK2 together with TTBK2 wt or TTBK2(KD) than in oocytes expressing GluK2 alone or together with either TTBK2(450) or TTBK2(450/KD). According to confocal microscopy of EGFP-tagged GluK2, TTBK2 wt decreased the GluK2 protein abundance in the cell membrane. Overexpression of an inactive RAB5(N133I) mutant but not RAB5wt could reverse the TTBK2 effect on GluK2 suggesting that RAB5 function is required for the effect. Conclusions: TTBK2 down-regulates GluK2 activity by decreasing the receptor protein abundance in the cell membrane via RAB5-dependent endocytosis, an effect that may protect against neuroexcitotoxicity.

Changes in microtubule-associated protein tau during peripheral nerve injury and regeneration.

Tau, a primary component of microtubule-associated protein, promotes microtubule assembly and/or disassembly and maintains the stability of the microtubule structure. Although the importance of tau in neurodegenerative diseases has been well demonstrated, whether tau is involved in peripheral nerve regeneration remains unknown. In the current study, we obtained sciatic nerve tissue from adult rats 0, 1, 4, 7, and 14 days after sciatic nerve crush and examined tau mRNA and protein expression levels and the location of tau in the sciatic nerve following peripheral nerve injury. The results from our quantitative reverse transcription polymerase chain reaction analysis showed that compared with the uninjured control sciatic nerve, mRNA expression levels for both tau and tau tubulin kinase 1, a serine/threonine kinase that regulates tau phosphorylation, were decreased following peripheral nerve injury. Our western blot assay results suggested that the protein expression levels of tau and phosphorylated tau initially decreased 1 day post nerve injury but then gradually increased. The results of our immunohistochemical labeling showed that the location of tau protein was not altered by nerve injury. Thus, these results showed that the expression of tau was changed following sciatic nerve crush, suggesting that tau may be involved in peripheral nerve repair and regeneration.

TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation.

Microtubules (MTs) play critical roles in various cellular events, including cell migration. End-binding proteins (EBs) accumulate at the ends of growing MTs and regulate MT end dynamics by recruiting other plus end-tracking proteins (+TIPs). However, how EBs contribute to MT dynamics through +TIPs remains elusive. We focused on tau-tubulin kinase 2 (TTBK2) as an EB1/3-binding kinase and confirmed that TTBK2 acted as a +TIP. We identified MT-depolymerizing kinesin KIF2A as a novel substrate of TTBK2. TTBK2 phosphorylated KIF2A at S135 in intact cells in an EB1/3-dependent fashion and inactivated its MT-depolymerizing activity in vitro. TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion. Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration. These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration.

Control of initial steps of ciliogenesis by protein kinases

Cilia are microtubule-based organelles present on the surface of most vertebrate cells. The formation of the primary cilia requires the mother centriole, which is the older of the two centrioles, to convert into the ciliary basal body. Although recent research has begun to shed light onto the molecular composition of the cilium, the regulation of ciliogenesis is just beginning to be understood. To get insight on the molecular mechanisms that regulate basal body formation, we performed a high-throughput RNA interference screen to identify protein kinases required for ciliogenesis. Of the novel kinases we identified, we are characterizing the function of the microtubule associated/affinity regulating kinase 4 (MARK4), and tau-tubulin kinase 2 (TTBK2). We show that MARK4 and TTBK2 are both required to initiate axoneme extension and to promote the removal of the inhibitory protein complex composed of CP110/Cep97. Together, our data indicate that cilia formation is a highly regulated process, which requires the concerted action of protein kinases that regulate the transition from the mother centriole into the basal body.

Tau tubulin kinases promote proteinopathy in both ftld-tau and FTLD-TDP-43

Tau tubulin kinases promote proteinopathy in both ftld-tau and FTLD-TDP-43

TTBK2: a tau protein kinase beyond tau phosphorylation.

Tau tubulin kinase 2 (TTBK2) is a kinase known to phosphorylate tau and tubulin. It has recently drawn much attention due to its involvement in multiple important cellular processes. Here, we review the current understanding of TTBK2, including its sequence, structure, binding sites, phosphorylation substrates, and cellular processes involved. TTBK2 possesses a casein kinase 1 (CK1) kinase domain followed by a ~900 amino acid segment, potentially responsible for its localization and substrate recruitment. It is known to bind to CEP164, a centriolar protein, and EB1, a microtubule plus-end tracking protein. In addition to autophosphorylation, known phosphorylation substrates of TTBK2 include tau, tubulin, CEP164, CEP97, and TDP-43, a neurodegeneration-associated protein. Mutations of TTBK2 are associated with spinocerebellar ataxia type 11. In addition, TTBK2 is essential for regulating the growth of axonemal microtubules in ciliogenesis. It also plays roles in resistance of cancer target therapies and in regulating glucose and GABA transport. Reported sites of TTBK2 localization include the centriole/basal body, the midbody, and possibly the mitotic spindles. Together, TTBK2 is a multifunctional kinase involved in important cellular processes and demands augmented efforts in investigating its functions.