Tau Tubulin Kinase Research Articles

Computational Design of Novel Tau-Tubulin Kinase 1 Inhibitors for Neurodegenerative Diseases.

The tau-tubulin kinase 1 (TTBK1) protein is a casein kinase 1 superfamily member located at chromosome 6p21.1. It is expressed explicitly in the brain, particularly in the cytoplasm of cortical and hippocampal neurons. TTBK1 has been implicated in the phosphorylation and aggregation of tau in Alzheimer's disease (AD). Considering its significance in AD, TTBK1 has emerged as a promising target for AD treatment. In the present study, we identified novel TTBK1 inhibitors using various computational techniques. We performed a virtual screening-based docking study followed by E-pharmacophore modeling, cavity-based pharmacophore, and ligand design techniques and found ZINC000095101333, LD7, LD55, and LD75 to be potential novel TTBK1 lead inhibitors. The docking results were complemented by Molecular Mechanics/Generalized Born Surface Area (MMGBSA) calculations. The molecular dynamics (MD) simulation studies at a 500 ns scale were carried out to monitor the behavior of the protein toward the identified ligands. Pharmacological and ADME/T studies were carried out to check the drug-likeness of the compounds. In summary, we identified a new series of compounds that could effectively bind the TTBK1 receptor. The newly designed compounds are promising candidates for developing therapeutics targeting TTBK1 for AD.

Regulation of primary cilia disassembly through HUWE1-mediated TTBK2 degradation plays a crucial role in cerebellar development and medulloblastoma growth

Development of the cerebellum requires precise regulation of granule neuron progenitor (GNP) proliferation. Although it is known that primary cilia are necessary to support GNP proliferation, the exact molecular mechanism governing primary cilia dynamics within GNPs remains elusive. Here, we establish the pivotal roles for the centrosomal kinase TTBK2 (Tau tubulin kinase-2) and the E3 ubiquitin ligase HUWE1 in GNP proliferation. We show that TTBK2 is highly expressed in proliferating GNPs under Sonic Hedgehog (SHH) signaling, coinciding with active GNP proliferation and the presence of primary cilia. TTBK2 stabilizes primary cilia by inhibiting their disassembly, thereby promoting GNP proliferation in response to SHH. Mechanistically, we identify HUWE1 as a novel centrosomal E3 ligase that facilitates primary cilia disassembly by targeting TTBK2 degradation. Disassembly of primary cilia serves as a trigger for GNP differentiation, allowing their migration from the external granule layer (EGL) of the cerebellum to the internal granule layer (IGL) for subsequent maturation. Moreover, we have established a link between TTBK2 and SHH-type medulloblastoma (SHH-MB), a tumor characterized by uncontrolled GNP proliferation. TTBK2 depletion inhibits SHH-MB proliferation, indicating that TTBK2 may be a potential therapeutic target for this cancer type. In summary, our findings reveal the mechanism governing cerebellar development and highlight a potential anti-cancer strategy for SHH-MB.

Liraglutide versus pramlintide in protecting against cognitive function impairment through affecting PI3K/AKT/GSK-3β/TTBK1 pathway and decreasing Tau hyperphosphorylation in high-fat diet- streptozocin rat model.

The American Diabetes Association guidelines (2021) confirmed the importance of raising public awareness of diabetes-induced cognitive impairment, highlighting the links between poor glycemic control and cognitive impairment. The characteristic brain lesions of cognitive dysfunction are neurofibrillary tangles (NFT) and senile plaques formed of amyloid-β deposition, glycogen synthase kinase 3 beta (GSK3β), and highly homologous kinase tau tubulin kinase 1 (TTBK1) can phosphorylate Tau proteins at different sites, overexpression of these enzymes produces extensive phosphorylation of Tau proteins making them insoluble and enhance NFT formation, which impairs cognitive functions. The current study aimed to investigate the potential contribution of liraglutide and pramlintide in the prevention of diabetes-induced cognitive dysfunction and their effect on the PI3K/AKT/GSK-3β/TTBK1 pathway in type 2 diabetic (T2D) rat model. T2D was induced by administration of a high-fat diet for 10weeks, then injection of a single dose of streptozotocin (STZ); treatment was started with either pramlintide (200μg/kg/day sc) or liraglutide (0.6mg/kg/day sc) for 6weeks in addition to the HFD. At the end of the study, cognitive functions were assessed by novel object recognition and T-maze tests. Then, rats were sacrificed for biochemical and histological assessment of the hippocampal tissue. Both pramlintide and liraglutide treatment revealed equally adequate control of diabetes, prevented the decline in memory function, and increased PI3K/AKT expression while decreasing GSK-3β/TTBK1 expression; however, liraglutide significantly decreased the number of Tau positive cells better than pramlintide did. This study confirmed that pramlintide and liraglutide are promising antidiabetic medications that could prevent associated cognitive disorders in different mechanisms.

Illumination of understudied ciliary kinases.

Cilia are cellular signaling hubs. Given that human kinases are central regulators of signaling, it is not surprising that kinases are key players in cilia biology. In fact, many kinases modulate ciliogenesis, which is the generation of cilia, and distinct ciliary pathways. Several of these kinases are understudied with few publications dedicated to the interrogation of their function. Recent efforts to develop chemical probes for members of the cyclin-dependent kinase like (CDKL), never in mitosis gene A (NIMA) related kinase (NEK), and tau tubulin kinase (TTBK) families either have delivered or are working toward delivery of high-quality chemical tools to characterize the roles that specific kinases play in ciliary processes. A better understanding of ciliary kinases may shed light on whether modulation of these targets will slow or halt disease onset or progression. For example, both understudied human kinases and some that are more well-studied play important ciliary roles in neurons and have been implicated in neurodevelopmental, neurodegenerative, and other neurological diseases. Similarly, subsets of human ciliary kinases are associated with cancer and oncological pathways. Finally, a group of genetic disorders characterized by defects in cilia called ciliopathies have associated gene mutations that impact kinase activity and function. This review highlights both progress related to the understanding of ciliary kinases as well as in chemical inhibitor development for a subset of these kinases. We emphasize known roles of ciliary kinases in diseases of the brain and malignancies and focus on a subset of poorly characterized kinases that regulate ciliary biology.

Computational identification of potential tau tubulin kinase 1 (TTBK1) inhibitors: a structural analog approach.

The online version contains supplementary material available at 10.1007/s40203-024-00242-z.

Antisense oligonucleotide-based targeting of Tau-tubulin kinase 1 prevents hippocampal accumulation of phosphorylated tau in PS19 tauopathy mice

Tau tubulin kinase-1 (TTBK1), a neuron-specific tau kinase, is highly expressed in the entorhinal cortex and hippocampal regions, where early tau pathology evolves in Alzheimer’s disease (AD). The protein expression level of TTBK1 is elevated in the cortex brain tissues with AD patients compared to the control subjects. We therefore hypothesized that antisense oligonucleotide (ASO) based targeting Ttbk1 could prevent the accumulation of phosphorylated tau, thereby delaying the development of tau pathology in AD. Here we show that in vivo administration of ASO targeting mouse Ttbk1 (ASO-Ttbk1) specifically suppressed the expression of Ttbk1 without affecting Ttbk2 expression in the temporal cortex of PS19 tau transgenic mice. Central administration of ASO-Ttbk1 in PS19 mice significantly reduced the expression level of representative phosphor-tau epitopes relevant to AD at 8 weeks post-dose, including pT231, pT181, and pS396 in the sarkosyl soluble and insoluble fractions isolated from hippocampal tissues as determined by ELISA and pS422 in soluble fractions as determined by western blotting. Immunofluorescence demonstrated that ASO-Ttbk1 significantly reduced pS422 phosphorylated tau intensity in mossy fibers region of the dentate gyrus in PS19 mice. RNA-sequence analysis of the temporal cortex tissue revealed significant enrichment of interferon-gamma and complement pathways and increased expression of antigen presenting molecules (Cd86, Cd74, and H2-Aa) in PS19 mice treated with ASO-Ttbk1, suggesting its potential effect on microglial phenotype although neurotoxic effect was absent. These data suggest that TTBK1 is an attractive therapeutic target to suppress TTBK1 without compromising TTBK2 expression and pathological tau phosphorylation in the early stages of AD.

Graph Theoretical Network Analysis and Pharmacoinformatics-Based Investigation of Bioactive Compounds from Semecarpus anacardium Linn. for Alzheimer’s Disease

Alzheimer’s disease is a highly prevalent disease that has affected over 50 million people worldwide. It is mainly caused by the continuous deposition of amyloid-[Formula: see text] peptide (A[Formula: see text]. Acetylcholinesterase (AChE), N-methyl D-aspartate (NMDA), Tau-Tubulin kinase 1 (TTBK1) and [Formula: see text]-secretase 1 (BACE-1) are potential targets to treat Alzheimer’s disease and to reduce the pathogenesis; amongst, BACE-1 is preferred owing to its capacity in minimizing the accumulation of A[Formula: see text]. This study explored Semecarpus anacardium Linn.-derived phytocompounds and their interaction with BACE-1. The phytochemicals of S. anacardiumLinn. were retrieved from the IMPPAT database. The factual targets (AChE, BACE-1, NMDA and TTBK1) of Alzheimer’s disease are identified by the graph theoretical network analysis, and are docked with a total of eight bioactive compounds from S. anacardium Linn. Interestingly, the docking score was significant (−12 kcal × mol[Formula: see text] for the interactions of amentoflavone with BACE-1. The co-crystallized ligand of BACE-1 (2-imino-3-methyl-5,5-diphenylimidazolidin-4-one) was considered for further studies and revealed a superior binding affinity (−8.7 kcal × mol[Formula: see text]. Molecular dynamics (MD) simulation and Density Functional Theory (DFT) analyzed the conformational stability of amentoflavone and BACE-1 complex. Furthermore, S. anacardium Linn. seemed to be nontoxic against U87-MG cells in vitro. These results from in silico prediction suggest that the derivatives of S. anacardium Linn. possess a significant interaction with the BACE-1 and provide a favorable direction to prevent aggregation of A[Formula: see text] peptides.

A Novel TTBK2 Mutation in a Chinese Pedigree with Spinocerebellar Ataxia 11.

Spinocerebellar ataxia type 11 (SCA11) is a rare disease and the tau tubulin kinase 2 (TTBK2) gene was the causative gene. To date, only six SCA11 families have been reported. Here, we reported a Chinese SCA11 pedigree with cerebellar ataxia. Both patients in the family demonstrated typical clinical features of cerebellar ataxia and cerebellar atrophy on brain MRI. A novel heterozygous duplication mutation (c.1211_1217dupAGGAGAA) of the TTBK2 gene was identified in the proband using whole-exome sequencing (WES), which resulted in a frameshift mutation and formed a premature stop codon (p. N406Kfs*47). The mutation was detected in the proband's affected brother, and his unaffected mother, who with a lower percentage of the mutation and considered as an asymptomatic mutation carrier. Our study delineated the genotypic spectrum of SCA11.

Tau tubulin kinase 1 and 2 regulate ciliogenesis and human pluripotent stem cells–derived neural rosettes

Primary cilia are key regulators of embryo development and tissue homeostasis. However, their mechanisms and functions, particularly in the context of human cells, are still unclear. Here, we analyzed the consequences of primary cilia modulation for human pluripotent stem cells (hPSCs) proliferation and differentiation. We report that neither activation of the cilia-associated Hedgehog signaling pathway nor ablation of primary cilia by CRISPR gene editing to knockout Tau Tubulin Kinase 2 (TTBK2), a crucial ciliogenesis regulator, affects the self-renewal of hPSCs. Further, we show that TTBK1, a related kinase without previous links to ciliogenesis, is upregulated during hPSCs-derived neural rosette differentiation. Importantly, we demonstrate that while TTBK1 fails to localize to the mother centriole, it regulates primary cilia formation in the differentiated, but not the undifferentiated hPSCs. Finally, we show that TTBK1/2 and primary cilia are implicated in the regulation of the size of hPSCs-derived neural rosettes.

Fragment-based screening approach reveals non-orthosteric pockets in the search for allosteric inhibitors of tau-tubulin kinase 1

Fragment-based screening approach reveals non-orthosteric pockets in the search for allosteric inhibitors of tau-tubulin kinase 1

Identification of tau-tubulin kinase 1 inhibitors by microfluidics-based mobility shift assay from a kinase inhibitor library

Tau tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that phosphorylates multiple residues in tau protein. Hyperphosphorylated tau is the main cause of tauopathy, such as Alzheimer's disease (AD). Therefore, preventing tau phosphorylation by inhibiting TTBK1 has been proposed as a therapeutic strategy for AD. However, few substrates of TTBK1 are reported for a biochemical assay and few inhibitors targeting TTBK1 have been reported so far. In this study, we identified a fluorescein amidite (FAM)-labeled peptide 15 from a small peptide library as the optimal peptide substrate for human TTBK1 (hTTBK1). We then developed and validated a microfluidics-based mobility shift assay (MMSA) with peptide 15. We further confirmed that peptide 15 could also be used in the ADP-Glo kinase assay. The established MMSA was applied for screening of a 427-compound kinase inhibitor library, yielding five compounds with IC50s of several micro molars against hTTBK1. Among them, three compounds, AZD5363, A-674,563 and GSK690693 inhibited hTTBK1 in an ATP competitive manner and molecular docking simulations revealed that they enter the ATP pocket and form one or two hydrogen bonds to the hinge region with hTTBK1. Another hit compound, piceatannol, showed non-ATP competitive inhibitory effect on hTTBK1 and may serve as a starting point to develop highly selective hTTBK1 inhibitors. Altogether, this study provided a new in vitro platform for the development of novel hTTBK1 inhibitors that might have potential applications in AD prevention.

Spinocerebellar ataxia type 11 (SCA11): An update.

Spinocerebellar ataxias, also called autosomal dominant cerebellar ataxias, are a group of neurological genetic diseases characterised by chronic, progressive cerebellar ataxia. The clinical hallmark of spinocerebellar ataxia is the loss of balance and coordination, accompanied by slurred speech. Spinocerebellar ataxia type 11 is a rare subtype of spinocerebellar ataxia caused by mutations in the tau tubulin kinase 2 gene. Patients with spinocerebellar ataxia are clinically characterised by slowly progressive cerebellar ataxia, trunk and limb ataxia, and eye movement abnormalities with occasional pyramidal features. Peripheral neuropathy and dystonia are rare. According to the literature, only nine families affected with spinocerebellar ataxia have been reported worldwide. Herein, a series of spinocerebellar ataxia cases are discussed in detail to determine the potential research direction of this dysfunction, including its epidemiology, clinical features, genetic characteristics, diagnosis and differential diagnosis, pathogenic mechanisms, treatment, prognosis, follow-up, genetic counselling and future perspectives, and to improve the overall understanding of spinocerebellar ataxia among clinicians, researchers and patients.

Modulation of tau tubulin kinases (TTBK1 and TTBK2) impacts ciliogenesis

Tau tubulin kinase 1 and 2 (TTBK1/2) are highly homologous kinases that are expressed and mediate disease-relevant pathways predominantly in the brain. Distinct roles for TTBK1 and TTBK2 have been delineated. While efforts have been devoted to characterizing the impact of TTBK1 inhibition in diseases like Alzheimer’s disease and amyotrophic lateral sclerosis, TTBK2 inhibition has been less explored. TTBK2 serves a critical function during cilia assembly. Given the biological importance of these kinases, we designed a targeted library from which we identified several chemical tools that engage TTBK1 and TTBK2 in cells and inhibit their downstream signaling. Indolyl pyrimidinamine 10 significantly reduced the expression of primary cilia on the surface of human induced pluripotent stem cells (iPSCs). Furthermore, analog 10 phenocopies TTBK2 knockout in iPSCs, confirming a role for TTBK2 in ciliogenesis.

Identification of Novel Tau-Tubulin Kinase 2 Inhibitors Using Computational Approaches.

Tau tubulin kinase 2 (TTBK2) associated with multiple diseases is one of the kinases which phosphorylates tau and tubulin. Numerous efforts have been made to understand the role of TTBK2 in protein folding mechanisms and misfolding behavior. The misfolded protein intermediates form polymers with unwanted aggregation properties that initiate several diseases, including Alzheimer's. The availability of TTBK2 inhibitors can enhance the understanding of the molecular mechanism of action of the kinase and assist in developing novel therapeutics. In the quest for TTBK2 inhibitors, this study focuses on screening two chemical libraries (ChEMBL and ZINC-FDA). The molecular docking, RO5/absorption, distribution, metabolism, and excretion/toxicity, density functional theory, molecular dynamics (MD) simulations, and molecular mechanics with generalized Born and surface area solvation techniques enabled shortlisting of the four most active compounds, namely, ChEMBL1236395, ChEMBL2104398, ChEMBL3427435, and ZINC000000509440. Moreover, 500 ns MD simulation was performed for each complex, which provided valuable insights into the structural changes in the complexes. The relative fluctuation, solvent accessible surface area, atomic gyration, compactness covariance, and free energy landscapes revealed that the compounds could stabilize the TTBK2 protein. Overall, this study would be valuable for the researchers targeting the development of novel TTBK2 inhibitors.

Spinocerebellar ataxia type 11 (SCA11): TTBK2 variants, functions and associated disease mechanisms.

Spinocerebellar ataxia type 11 (SCA11) is a rare type of autosomal dominant cerebellar ataxia, mainly characterized by progressive cerebellar ataxia, abnormal eye signs and dysarthria. SCA11 is caused by variants in TTBK2, which encodes tau tubulin kinase 2 (TTBK2) protein. Only a few families with SCA11 were described to date, all harbouring small deletions or insertions that result in frameshifts and truncated TTBK2 proteins. In addition, TTBK2 missense variants were also reported but they were either benign or still needed functional validation to ascertain their pathogenic potential in SCA11. The mechanisms behind cerebellar neurodegeneration mediated by TTBK2 pathogenic alleles are not clearly established. There is only one neuropathological report and a few functional studies in cell or animal models published to date. Moreover, it is still unclear whether the disease is caused by TTBK2 haploinsufficiency of by a dominant negative effect of TTBK2 truncated forms on the normal allele. Some studies point to a lack of kinase activity and mislocalization of mutated TTBK2, while others reported a disruption of normal TTBK2 function caused by SCA11 alleles, particularly during ciliogenesis. Although TTBK2 has a proven function in cilia formation, the phenotype caused by heterozygous TTBK2 truncating variants are not clearly typical of ciliopathies. Thus, other cellular mechanisms may explain the phenotype seen in SCA11. Neurotoxicity caused by impaired TTBK2 kinase activity against known neuronal targets, such as tau, TDP-43, neurotransmitter receptors or transporters, may contribute to neurodegeneration in SCA11.

Molecular hydrogen attenuates sepsis-induced cognitive dysfunction through regulation of tau phosphorylation.

Sepsis-associated encephalopathy (SAE) is a cognitive dysfunction caused by sepsis. Hyperphosphorylated tau is considered to play a significant role in the progression of neurodegenerative disease and also contributes to cognitive dysfunction in septic mice. Molecular hydrogen (H2) plays an antioxidant and anti-inflammatory role, and plays a protective role in septic mice. This study explored the possible effects of H2 on cognition and tau phosphorylation in a mouse model of SAE. The model of sepsis was established in C57BL/6J male mice by cecal ligation and puncture surgery. Mice treated with 2% H2 inhalation for 60min at 1h and 6h after surgery, respectively. HY-15769, the inhibitor of Tau Tubulin Kinase 1 (TTBK1), was injected 1h before the surgery. The 7-day survival rates of the mice were recorded. Cognitive behavior was tested with both novel object recognition and the Y-maze novelty arm recognition on day 7 after surgery. Hematoxylin-eosin staining was used to observe the histological damage in CA1 region of hippocampus. The expression of inflammatory factors in hippocampus was assessed by Elisa. Western blotting was adopted to determine the tau phosphorylation levels at AT8 epitopes (pSer202 and pThr205) and T22 epitopes (neurofibrillary tangle protein oligomer), and the GSK3β phosphorylation levels (Tyr216), as well as p-Ser422 and TTBK1 levels in the hippocampus. The number of dendritic spine and mushroom type of dendritic spines in the hippocampus were assessed by Golgi staining. The survival rate, visual and spatial learning ability, and memory ability were improved in septic mice treated with H2. After H2 treatment, the density of dendritic spine, mushroom type of dendritic spine, and the number of normal hippocampal neurons were progressively elevated. H2 decreased the levels of phosphorylated tau protein, tau oligomer and TTBK1, as well as the phosphorylation of tau key kinase. Furthermore, the injection of HY-15769 (a TTBK1 inhibitor) protected SAE through the similar way. The protective effect of H2 on cognitive dysfunction induced by SAE may be achieved by inhibiting tau phosphorylation, which is perhaps related with the inhibition of TTBK1.

TTBK2 controls cilium stability by regulating distinct modules of centrosomal proteins.

The serine-threonine kinase tau tubulin kinase 2 (TTBK2) is a key regulator of the assembly of primary cilia, which are vital signaling organelles. TTBK2 is also implicated in the stability of the assembled cilium through mechanisms that remain to be defined. Here we use mouse embryonic fibroblasts derived from Ttbk2fl/fl, UBC-CreERT+ embryos (hereafter Ttbk2cmut) to dissect the role of TTBK2 in cilium stability. This system depletes TTBK2 levels after cilia formation, allowing us to assess the molecular changes to the assembled cilium over time. As a consequence of Ttbk2 deletion, the ciliary axoneme is destabilized and primary cilia are lost within 48-72 h following recombination. Axoneme destabilization involves an increased frequency of cilia breaks and a reduction in axonemal microtubule modifications. Cilia loss was delayed by using inhibitors that affect actin-based trafficking. At the same time, we find that TTBK2 is required to regulate the composition of the centriolar satellites and to maintain the basal body pools of intraflagellar transport proteins. Altogether, our results reveal parallel pathways by which TTBK2 maintains cilium stability.

CK2 and protein kinases of the CK1 superfamily as targets for neurodegenerative disorders.

Casein kinases are involved in a variety of signaling pathways, and also in inflammation, cancer, and neurological diseases. Therefore, they are regarded as potential therapeutic targets for drug design. Recent studies have highlighted the importance of the casein kinase 1 superfamily as well as protein kinase CK2 in the development of several neurodegenerative pathologies, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. CK1 kinases and their closely related tau tubulin kinases as well as CK2 are found to be overexpressed in the mammalian brain. Numerous substrates have been detected which play crucial roles in neuronal and synaptic network functions and activities. The development of new substances for the treatment of these pathologies is in high demand. The impact of these kinases in the progress of neurodegenerative disorders, their bona fide substrates, and numerous natural and synthetic compounds which are able to inhibit CK1, TTBK, and CK2 are discussed in this review.

TDP-43 Modulation by Tau-Tubulin Kinase 1 Inhibitors: A New Avenue for Future Amyotrophic Lateral Sclerosis Therapy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease without any effective treatment. Protein TDP-43 is a pathological hallmark of ALS in both sporadic and familiar patients. Post-translational modifications of TDP-43 promote its aggregation in the cytoplasm. Tau-Tubulin kinase (TTBK1) phosphorylates TDP-43 in cellular and animal models; thus, TTBK1 inhibitors emerge as a promising therapeutic strategy for ALS. The design, synthesis, biological evaluation, kinase-ligand complex structure determination, and molecular modeling studies confirmed novel pyrrolopyrimidine derivatives as valuable inhibitors for further development. Moreover, compound 29 revealed good brain penetration in vivo and was able to reduce TDP-43 phosphorylation not only in cell cultures but also in the spinal cord of transgenic TDP-43 mice. A shift to M2 anti-inflammatory microglia was also demonstrated in vivo. Both these activities led to motor neuron preservation in mice, proposing pyrrolopyrimidine 29 as a valuable lead compound for future ALS therapy.

Tau-tubulin kinase 1 phosphorylates TDP-43 at disease-relevant sites and exacerbates TDP-43 pathology

TDP-43 pathology is a hallmark of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal lobar degeneration (FTLD). Namely, both diseases feature aggregated and phosphorylated TDP-43 containing inclusions in the cytoplasm and a loss of nuclear TDP-43 in affected neurons. It has been reported that tau tubulin kinase (TTBK)1/2 phosphorylate TDP-43 and TTBK1/2 overexpression induced neuronal loss and behavioral deficits in a C. elegans model of ALS. Here we aimed to elucidate the molecular mechanisms of TTBK1 in TDP-43 pathology. TTBK1 levels were observed to be elevated in ALS patients' post-mortem motor cortex. Also, TTBK1 was found to phosphorylate TDP-43 at disease-relevant sites in vitro directly, and this phosphorylation accelerated TDP-43 formation of high molecular species. Overexpression of TTBK1 in mammalian cells induced TDP-43 phosphorylation and the construction of high molecular species, concurrent with TDP-43 mis-localization and cytoplasmic inclusions. In addition, when TTBK1 was knocked down or pharmacologically inhibited, TDP-43 phosphorylation and aggregation were significantly alleviated. Functionally, TTBK1 knockdown could rescue TDP-43 overexpression-induced neurite and neuronal loss in iPSC-derived GABAergic neurons. These findings suggest that phosphorylation plays a critical role in the pathogenesis of TDP-43 pathology and that TTBK1 inhibition may have therapeutic potential for the treatment of ALS and FTLD.