TANK-binding Kinase 1 Gene Research Articles

Molecular characterization, expression, and functional identification of TANK-binding kinase 1 (TBK1) of the cow (Bos taurus) and goat (Capra hircus)

The role of TANK-binding kinase 1 (TBK1) of humans and mice in innate immunity is well elucidated. Still, the molecular characterization and biological function of the TBK1 gene in herbivorous animals are less studied. Here, the open reading frame (ORF) of TBK1 of the cow and goat was firstly cloned and successfully expressed. The Phylogenetic tree analysis reveals that the TBK1 gene of goats and cows is similar to chicken and mute swans, respectively. Some evolutionary distances of the TBK1 gene were still present among different species. A slightly subcellular distribution difference was observed among full-length and truncated TBK1 of goats and cows. Dual-luciferase reporter assay has shown that the full-length TBK1 of goats and cows plays a vital role in the induction of IFN-β production. The viral infection experiment showed that the over-expression of the full-length TBK1 gene of the cow and goat significantly suppresses intracellular viral replication of the Lumpy skin disease virus (LSDV) in infected cells. Our study showed that TBK1 in the cows and goats is a crucial immunoregulatory for IFN-β production during viral infection, contributing to a better understanding of innate immunity in the herbivorous animal.

Corticobasal syndrome associated with a novel Lys694del variant of the TBK1 gene.

Variants of the TANK-Binding Kinase 1 (TBK1) gene have been associated with frontotemporal dementia - amyotrophic lateral sclerosis (FTD-ALS) spectrum diseases. Corticobasal syndrome (CBS), characterized by asymmetric limb rigidity, dystonia or myoclonus, in association with speech or limb apraxia, cortical sensory deficit, and/or alien limb can result from a variety of underlying pathologies. It is most commonly sporadic, although it has been rarely described in association with genetic defects, especially MAPT and GRN genes. Case study. We describe the proband of a family with multiple occurrences of FTD-ALS spectrum disease who presented with a primary asymmetric akinetic-rigid syndrome with later speech involvement and cognitive dysfunction, contralateral anterior temporal lobe atrophy on MRI and corresponding hypometabolism by FDG-PET, who carried a novel Lys694del variant of the TBK1 gene and predominant Type A TDP-43 pathology in a frontotemporal distribution that was contralateral to the affected side. Although CBS has been described as a late feature of agrammatic primary progressive aphasia (PPA) in a patient carrying a different variant TBK1, to our knowledge our case is the first report of a patient presenting with asymmetric akinetic rigidity as the initial expression of a TBK1 variant. This case provides further support to consider TBK1 genetic screening in patients with CBS and a family history suggestive of FTD-ALS spectrum diseases, as this may be an underrepresented population on the spectrum of genetic FTD-ALS.

Novel TBK1 variant associated with Frontotemporal Dementia overlap syndrome

Recently, TANK binding kinase 1 (TBK1) mutation has been reported as a causative gene for overlap frontotemporal dementia (FTD)-amyotrophic lateral sclerosis (ALS) syndrome. However, there are no reports from families of South Asian ethnicity.To report a case study of a family with the proband having overlap FTD-ALS syndrome caused by a novel TBK1 variant.Clinical, brain imaging, genetic analysis and laboratory data of the patient with FTD-ALS were performed. In addition, family-based segregation analysis of identified novel variants was also done.This study pertains to genetic analysis in 11 members in a family with only one member affected with overlap FTD-ALS syndrome. The whole-exome sequencing analysis in the symptomatic member showed a novel loss-of-function (LoF) variant c.1810G>T(p.E604X) in the TBK1 gene. Neuroimaging showed a pattern of asymmetric frontotemporal atrophy and hypometabolism. Segregation analysis of the variation demonstrated its presence in several family members, although none of the other members was symptomatic. Further, we observed another missense variation in the NEFH gene (p.Pro683Leu) which was seen in the symptomatic and two asymptomatic family members, the pathogenicity of which is unclear.This is the first study of a rare novel TBK1 variant associated with FTD-ALS from India. Asymptomatic family members with the variant have important clinical implications and necessitate the genetic evaluation and long-term follow-up of family members of patients detected with TBK1 mutations. Therefore, although infrequent, genetic screening for the TBK1 gene should be considered when encountering overlap FTD syndromes.

Long-Term Follow-Up of Normal Tension Glaucoma Patients With TBK1 Gene Mutations in One Large Pedigree

To characterize features of glaucoma associated with a TANK binding kinase 1 (TBK1) gene duplication, which is among the most common molecularly defined causes of normal tension glaucoma (NTG). Retrospective observational case series. We conducted a retrospective case series, by reviewing medical records of 7 members of a pedigree with NTG caused by TBK1 gene duplications. Clinical features of these patients at diagnosis, throughout management, and at latest follow-up were identified, including age, intraocular pressure (IOP), central corneal thickness (CCT), optic nerve head appearance, and mean deviation (MD) assessed with Humphrey visual field (HVF) testing protocols. At initial diagnosis, the mean age was 35 ± 7 years, IOP was 16 ± 2.1mm Hg, cup-to-disc (C/D) ratio was 0.9 ± 0.08, and MD assessed via HVF 30-2 and/or 24-2 testing protocols was-9.0 ± 8.9 (range:-1.8 to-27) dB in the 14 study eyes. At initial diagnosis, 4 of 14 eyes (28%) had no visual field defect, 4 (28%) had early visual field defects, and 6 (43%) had severe visual field defects. Patients had a mean follow-up of 21.5 ± 9.0 years and experienced an average reduction of IOP by 28%. Four of 12 eyes (33%) had stable visual fields throughout follow-up, while 8 eyes (67%) had slow-to-moderate progression. The 30-2 and/or 24-2 HVF tests had an average change in MD of-0.53 ± 0.26 dB/year. No eyes had rapid progression with an MD >1.0 dB/year. At final follow-up, the mean IOP was 11.5 ± 2.9, and C/D ratio was 0.94 ± 0.4. At final follow-up, 3 of 14 eyes (21%) had early visual field defects, 4 (29%) had moderate visual field defects, and 7 (50%) had severe visual field defects. Six of 14 eyes (43%) met criteria for legal blindness. We provide the first report of the clinical features and long-term clinical course in a family of NTG patients with TBK1 gene duplications. TBK1-associated glaucoma exhibits classic features of NTG. Patients present with severe disease at a relatively early age and most (67%) have slow-to-moderate progression of their visual field defects. The rate of visual field change appears correlated with the magnitude of IOP, suggesting that it may be advantageous to set extremely low IOP targets for some patients with TBK1-associated glaucoma.

Investigating the TANK-binding kinase expression in multiple sclerosis patients in comparison with control group in the Iranian population

Introduction: Multiple sclerosis (MS) is one of the most common inflammatory autoimmune diseases in the central nervous system, which affects individuals ranged between 20 and 40 years of age. There is still no gene or genes set that is unique to the disease. On the other hand, TANK-binding kinase 1 (TBK1) gene is one of the most important interferon signalling molecules and has been shown to affect many autoimmune diseases, and it is obvious that it is involved in CNS inflammation. In this study, we investigated the expression of TBK1 gene as a major factor in the production and activity of interferons in patients with MS. Materials and Methods: Ninety patients with MS and 30 healthy individuals referring to Kashani Hospital in Shahrekord which matched for age and sex were selected. According to the received treatments, MS patients were divided into three groups as follows: the newly diagnosed group not receiving treatment, interferon β1α (IFNβ1α) treatment group and IFNβ1β treatment group. After filling the questionnaire, blood was sampled, total RNA was extracted and finally, cDNA was synthesised from the individuals. The TBK1 kinase gene expression was investigated using the TaqMan real-time with ΔΔCt method. Obtained data were analysed using GRAPHPAD 5 and SPSS version 21 software. Results: In this study, the two studied groups did not show any significant differences in age and sex parameters. Our results showed that TBK1 expression in newly diagnosed patients was significantly higher than the control group (P

Retinoic acid worsens ATG10-dependent autophagy impairment in TBK1-mutant hiPSC-derived motoneurons through SQSTM1/p62 accumulation

ABSTRACT Mutations in the TBK1 (TANK binding kinase 1) gene are causally linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TBK1 phosphorylates the cargo receptors OPTN and SQSTM1 regulating a critical step in macroautophagy/autophagy. Disruption of the autophagic flux leads to accumulation of cytosolic protein aggregates, which are a hallmark of ALS. hiPSC-derived TBK1-mutant motoneurons (MNs) showed reduced TBK1 levels and accumulation of cytosolic SQSTM1-positive aggresomes. By screening a library of nuclear-receptor-agonists for modifiers of the SQSTM1 aggregates, we identified 4-hydroxy(phenyl)retinamide (4HPR) as a potent modifier exerting detrimental effects on mutant-TBK1 motoneurons fitness exacerbating the autophagy overload. We have shown by TEM that TBK1-mutant motoneurons accumulate immature phagophores due a failure in the elongation phase, and 4HPR further worsens the burden of dysfunctional phagophores. 4HPR-increased toxicity was associated with the upregulation of SQSTM1 in a context of strongly reduced ATG10, while rescue of ATG10 levels abolished 4HPR toxicity. Finally, we showed that 4HPR leads to a downregulation of ATG10 and to an accumulation of SQSTM1+ aggresomes also in hiPSC-derived C9orf72-mutant motoneurons. Our data show that cultured human motoneurons harboring mutations in TBK1 gene display typical ALS features, like decreased viability and accumulation of cytosolic SQSTM1-positive aggresomes. The retinoid 4HPR appears a strong negative modifier of the fitness of TBK1 and C9orf72-mutant MNs, through a pathway converging on the mismatch of initiated autophagy and ATG10 levels. Thus, autophagy induction appears not to be a therapeutic strategy for ALS unless the specific underlying pathway alterations are properly addressed. Abbreviations 4HPR: 4-hydroxy(phenyl)retinamide; AKT: AKT1 serine/threonine kinase 1; ALS: amyotrophic lateral sclerosis; ATG: autophagy related; AVs: autophagic vesicle; C9orf72: chromosome 9 open reading frame 72; CASP3: caspase 3; CHAT: choline O-acetyltransferase; CYCS: cytochrome c, somatic; DIV: day in vitro; FTD: frontotemporal dementia; FUS: FUS RNA binding protein; GFP: green fluorescent protein; hiPSCs: human induced pluripotent stem cells; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MNs: motoneurons; mRFP: monomeric red fluorescent protein; MTOR: mechanistic target of rapamycin kinase; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; RARA: retinoic acid receptor alpha; SLC18A3/VACHT: solute carrier family 18 (vesicular acetylcholine transporter), member 3; SQSTM1/p62: sequestosome 1; TBK1: TANK binding kinase 1; TEM: transmission electron microscopy

CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells.

TANK-binding kinase 1 (TBK1) is involved in innate immunity, prompting transcriptional induction of type I interferons in response to pathogenic infection. Many studies have focused on mammals but the function of TBK1 in chickens remains poorly defined. CRISPR/Cas9 system has made gene-knockout easy to accomplish. Although CRISPR/Cas9 has been used in chicken cells, low mutation efficiency limits its wide application in chickens. In this study, an effective gene-knockout system was developed based on the CRISPR/Cas9 system in chicken embryonic fibroblast DF-1. Two CRISPR/Cas9 plasmids were constructed, TBK1-g1 and TBK1-g2, which express gRNAs targeting different sequences of the chicken TBK1 gene. After transfection and enrichment with puromycin screening, the mutation rates as assessed via T7E1 assay were 88.05 and 89.55%, respectively, and subsequent sequence analysis showed mutation efficiencies of 86.67 and 93.33%. With the limiting-dilution method, a chTBK1 gene-deficiency monoclonal cell line was obtained and was named DF-1-TBK1-C3. The DF-1-TBK1-C3 cells exhibited normal morphology and maintained stable proliferation ability compared to wild-type cells. The gene-overexpression system and luciferase reporter assay showed that IFN-β induction induced by chSTING was almost completely blocked in DF-1-TBK1-C3 cells. With quantitative real-time PCR, we further confirmed the essential role of chTBK1 in the chSTING-mediated IFN-β induction. At last, the study demonstrated that the chTBK1 knockout system is also applicable in primary chick embryo fibroblasts (CEFs). In this study, an effective gene-knockout system was applied in chickens, a TBK1 gene-deleted DF-1 cell line was successfully created using this system, and with the chTBK1 knockout cells, chTBK1 was revealed to be indispensable in STING-mediated IFN-β activation in chicken cells.

Multi-Ethnic Comparison of the Characteristics of Amyotrophic Lateral Sclerosis-Related TBK1 Gene Variants

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease, and the etiology of sporadic ALS is unknown. The TANK-binding kinase 1 (TBK1) gene identified as an ALS gene, contributes towards a predisposition for ALS. In this review, we analyzed variants of TBK1 found in ALS cohort studies belonging to various regions and ethnic populations. TBK1 variants tend to be enriched in patients with ALS compared to patients without ALS. The frequency of TBK1 variants is more in the familial Caucasian than that in the Asian population. However, loss of function (LoF) variant associated with sporadic ALS is almost similar among the Asian group, including Japanese population. LoF variants were frequently reported to be associated with the TBK1 biology. These findings indicate that TBK1- LoF variants are pathogenic for ALS, regardless of race or region.

P36. 4-hydroxy(phenyl) retinamide (4HPR) worsens the pathological features of ALS-related TBK1-mutant motor neurons by further disrupting the autophagic flux at the elongation phase

TANK-binding kinase 1 (TBK1) is a protein playing a critical role in the activation of pro-survival pathways, by phosphorylating Akt, and in autophagy, as its phosphorylation and activation of the autophagic receptors p62 and optineurin is fundamental for the correct cargo recruitment at the forming autophagosome. Moreover, mutations in TBK1 gene have been recently linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In our study we aimed to uncover the molecular pathological mechanisms characterizing ALS patients carrying mutations in TBK1 gene. For this scope, we used as a model human induced Pluripotent Stem Cells (hiPSCs), and differentiated them into spinal motor neurons (MNs). TBK1-mutant MNs display reduced TBK1 protein levels up to 50%, supporting the theory that haploinsufficiency might be the pathogenic mechanism leading to ALS in these specific cases. Interestingly, as observed in post-mortem tissues, TBK1 MNs are characterized by decreased viability during long-term culture, and accumulate aberrant cytosolic aggresomes. In order to elucidate the impairment affecting the degrative pathway leading to aggresomes accumulation, and eventually circumvent the pathological phenotype, we performed a small molecule screening, which identified 4-hydroxy(phenyl) retinamide (4HPR) as a potent autophagy modifier. This synthetic retinoid failed in reverting the pathological phenotype in mutant motor neurons:in fact, TBK1-mutant cells accumulated immature phagophores when treated with 4HPR. This highlighted an impaired elongation phase in autophagy, further confirmed by the down regulation of several genes involved in this specific step of the degradative pathway. Notably, over expression of Atg10, a protein directly involved in the autophagic elongation phase, rescued the detrimental effect exerted by the retinoid. Finally, we demonstrated that 4HPR leads to a strong and faster accumulation of disease markers also in human C9ORF72-mutant motor neurons and pre-synthomatic SOD1G93A mice. Our results bring novel mechanistic insights into the autophagy impairment occurring in ALS, confirming that induction of the degratative pathway worsens the disease progression if the levels of the autophagy interplayers are not properly re-established.

Frequency and characteristics of the TBK1 gene variants in Japanese patients with sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease, and the etiology of sporadic ALS is generally unknown. The TANK-binding kinase 1 (TBK1) gene was identified as an ALS gene contributing to a predisposition toward ALS. To reveal the frequency and characteristics of variants of the TBK1 gene in sporadic ALS patients in Japan, we analyzed the TBK1 gene by exome sequencing in a large Japanese cohort of 713 sporadic ALS patients and 800 controls. We identified known or potentially toxic rare variants of TBK1 gene in 9 patients (1.26%) with sporadic ALS, including 4 novel missense variants (p.V23I, p.H322R, p.R358C, and p.T478I) and 3 loss-of-function variants (p.R357X, p.P378_I379del, and p.T419_G420del). The odds ratio between sporadic ALS patients and controls was 10.2 (p = 0.008, 95% confidence interval = 1.67–62.47). These findings support the contribution of TBK1 to the etiology of sporadic ALS in Japanese patients.

Genomic Organization of TBK1 Copy Number Variations in Glaucoma Patients.

Approximately 1% of normal tension glaucoma (NTG) cases are caused by TANK-binding kinase 1 (TBK1) gene duplications and triplications. However, the precise borders and orientation of these TBK1 gene copy number variations (CNVs) on chromosome 12 are unknown. We determined the exact borders of TBK1 CNVs and the orientation of duplicated or triplicated DNA segments in 5 NTG patients with different TBK1 mutations using whole-genome sequencing. Tandemly duplicated chromosome segments spanning the TBK1 gene were detected in 4 NTG patients, each with unique borders. Four of 5 CNVs had borders located within interspersed repetitive DNA sequences (Alu and long interspersed nuclear element-L1 elements), suggesting that mismatched homologous recombinations likely generated these CNVs. A fifth NTG patient had a complex rearrangement including triplication of a chromosome segment spanning the TBK1 gene. No specific mutation hotspots for TBK1 CNVs were detected, however, interspersed repetitive sequences (ie, Alu elements) were identified at the borders of TBK1 CNVs, which suggest that mismatch of these elements during meiosis may be the mechanism that generated TBK1 gene dosage mutations.

Association of the New Variant Tyr424Asp at TBK1 Gene with Amyotrophic Lateral Sclerosis and Cognitive Decline.

A new risk gene associated with amyotrophic lateral sclerosis (ALS) has recently been identified: the Tank-binding kinase 1 (TBK1) gene. Up to now, 90 TBK1 variants have been described in ALS patients with or without frontotemporal dementia (FTD), thus making TBK1 the third or fourth most frequent genetic cause of ALS and FTD. A point mutation analysis in a cohort of 69 Italian ALS patients was performed in order to analyze the frequency of TBK1 mutations and the correlation with clinical phenotypes. The analysis identified the novel variant p.Tyr424Asp in a patient with a rapid progression of the disease. Our data supports the implication of TBK1 in ALS pathogenesis in Italy.

The up-regulation of TANK-binding kinase 1 in head and neck squamous cell carcinoma

Background: The purpose of the present study was to investigate the function of TANK-binding kinase 1 (TBK1) in head and neck squamous cell carcinoma (HNSCC) tumorigenesis and progression, and to clarify that whether TBK1 could be a biomarker for early diagnosis or even therapeutic target of HNSCC. Methods: We first analyzed the expression level of TBK1 mRNA in HNSCC using GEO datasets, and then the results were verified by tissue microarray (TMA). Finally, we suppressed TBK1 gene expression in FaDu and Cal-27 cells to investigate the cellular mechanism. Results: Results of GEO datasets and TMA showed that the expression level of TBK1 in HNSCC was higher than normal tissue. In FaDu/siTBK1 and Cal-27/siTBK1 cells, TBK1 protein expression was down regulated significantly and the proliferation rate decreased with apoptosis increased. Conclusions: TBK1 gene expression was important for HNSCC cell proliferation and might offer additional help for the early diagnosis of HNSCC and it might even be a meaningful therapeutic target, however the mechanisms and applicability still need further investigation.

Molecular characterization, expression of chicken TBK1 gene and its effect on IRF3 signaling pathway.

TRAF family member-associated NF-κB activator (TANK)-binding kinase1 (TBK1) is a serine-threonine kinase at the crossroads of multiple interferon (IFN)-inducing signaling pathways in innate immunity. The importance of TBK1 in antiviral immunity is well established in mammal models, but in chicken, the molecular characterization and potential function of TBK1 remain unclear. In the present study, the open-reading frame (ORF) of chicken TBK1 (chTBK1) was cloned and characterized. The sequencing results revealed that the chTBK1 ORF consists of 2190 base pairs (bp) encoding a deduced protein of 729 amino acid residues. Multiple sequence alignment analysis demonstrated chTBK1 similarity to other birds and mammals, which indicates that it is evolutionarily conserved. Quantitative real-time PCR (qRT-PCR) results showed that chTBK1 was ubiquitously expressed in chicken tissues and expression was especially high in immune tissues. In addition, the expression of chTBK1 was significantly up-regulated by infection with avian leukosis virus subgroup J (ALV-J) both in vivo and in chicken embryo fibroblasts (CEFs) challenged with ALV-J or stimulated with poly I:C in vitro. Consistent with the activation of chTBK1, the interferon regulatory factor 3 (IRF3) and IFNβ gene in CEFs were also up-regulated after challenge with ALV-J or polyI:C. In contrast, the expression of IRF3 and IFNβ in CEFs was significantly reduced by siRNA targeting the chTBK1 gene compared with a negative control (NC) during ALV-J infection or polyI:C transfection. In conclusion, our results demonstrated that chTBK1 may be an important immunoregulator for IRF3 and IFNβ induction in response to viral stimulation in chicken.

The clinical, neuroanatomical, and neuropathologic phenotype of TBK1-associated frontotemporal dementia: A longitudinal case report

IntroductionMutations in the TANK-binding kinase 1 (TBK1) gene have recently been shown to cause frontotemporal dementia (FTD). However, the phenotype of TBK1-associated FTD is currently unclear. MethodsWe performed a single case longitudinal study of a patient who was subsequently found to have a novel A705fs mutation in the TBK1 gene. He was assessed annually over a 7-year period with a series of clinical, cognitive, and magnetic resonance imaging assessments. His brain underwent pathological examination at postmortem. ResultsThe patient presented at the age of 64 years with an 18-month history of personality change including increased rigidity and obsessiveness, apathy, loss of empathy, and development of a sweet tooth. His mother had developed progressive behavioral and cognitive impairment from the age of 57 years. Neuropsychometry revealed intact cognition at first assessment. Magnetic resonance imaging showed focal right temporal lobe atrophy. Over the next few years his behavioral problems progressed and he developed cognitive impairment, initially with anomia and prosopagnosia. Neurological examination remained normal throughout without any features of motor neurone disease. He died at the age of 72 years and postmortem showed TDP-43 type A pathology but with an unusual novel feature of numerous TAR DNA-binding protein 43 (TDP-43)–positive neuritic structures at the cerebral cortex/subcortical white matter junction. There was also associated argyrophilic grain disease not previously reported in other TBK1 mutation cases. DiscussionTBK1-associated FTD can be associated with right temporal variant FTD with progressive behavioral change and relatively intact cognition initially. The case further highlights the benefits of next-generation sequencing technologies in the diagnosis of neurodegenerative disorders and the importance of detailed neuropathologic analysis.

TANK-binding kinase-1 broadly affects oyster immune response to bacteria and viruses.

As a benthic filter feeder of estuaries, the immune system of oysters provides one of the best models for studying the genetic and molecular basis of the innate immune pathway in marine invertebrates and examining the influence of environmental factors on the immune system. Here, the molecular function of molluscan TANK-binding kinase-1 (TBK1) (which we named CgTBK1) was studied in the Pacific oyster, Crassostrea gigas. Compared with known TBK1 proteins in other model organisms, CgTBK1 contains a conserved S-TKc domain and a coiled coil domain at the N- and C-terminals but lacks an important ubiquitin domain. Quantitative real-time PCR analysis revealed that the expression level of CgTBK1 was ubiquitous in all selected tissues, with highest expression in the gills. CgTBK1 expression was significantly upregulated in response to infections with Vibrio alginolyticus, ostreid herpesvirus 1 (OsHV-1 reference strain and μvar), and polyinosinic:polycytidylic acid sodium salt, suggesting its broad function in immune response. Subcellular localization showed the presence of CgTBK1 in the cytoplasm of HeLa cells, suggesting its potential function as the signal transducer between the receptor and transcription factor. We further demonstrated that CgTBK1 interacted with CgSTING in HEK293T cells, providing evidence that CgTBK1 could be activated by direct binding to CgSTING. In summary, we characterized the TBK1 gene in C.gigas and demonstrated its role in the innate immune response to pathogen infections.

Cutting Edge: Inhibiting TBK1 by Compound II Ameliorates Autoimmune Disease in Mice.

TANK-binding kinase 1 (TBK1) is a serine/threonine protein kinase that plays a crucial role in innate immunity. Enhanced TBK1 function is associated with autoimmune diseases and cancer, implicating the potential benefit of therapeutically targeting TBK1. In this article, we examined a recently identified TBK1 inhibitor Compound II on treating autoimmune diseases. We found that Compound II is a potent and specific inhibitor of TBK1-mediated IFN response. Compound II inhibited polyinosinic-polycytidylic acid-induced immune activation in vitro and in vivo. Compound II treatment also ameliorated autoimmune disease phenotypes of Trex1(-/-) mice, increased mouse survival, and dampened the IFN gene signature in TREX1 mutant patient lymphoblasts. In addition, we found that TBK1 gene expression is elevated in systemic lupus erythematosus patient cells, and systemic lupus erythematosus cells with high IFN signature responded well to Compound II treatment. Together, our findings provided critical experimental evidence for inhibiting TBK1 with Compound II as an effective treatment for TREX1-associated autoimmune diseases and potentially other interferonopathies.

TBK1Gene Duplication and Normal-Tension Glaucoma

Normal-tension glaucoma (NTG) is a common cause of vision loss. To investigate the role of TANK binding kinase 1 (TBK1) gene duplications in NTG to gain insights into the causes of glaucoma that occurs at low intraocular pressure (IOP). In this multicenter case-control study, we investigated patients who met the criteria for NTG, including glaucomatous optic neuropathy, visual field defects, and maximum recorded untreated IOP of 21 mm Hg or less, and matched controls. Participants (N = 755) were recruited from Southampton, United Kingdom (180 patients and 178 controls), Rochester, Minnesota (65 patients and 12 controls), New York, New York (96 patients and 16 controls), and Iowa City, Iowa (208 controls). Detection of TBK1 gene duplications and comparison of the extent of the identified DNA that is duplicated with prior TBK1 copy number variations associated with NTG. A TBK1 gene duplication was detected in 1 of 96 patients (1.0%) from New York and none of the controls. Analysis of duplication borders with comparative genome hybridization demonstrated that this patient has a novel duplication that has not been previously reported. No gene duplications were detected in any of the other cohorts of patients or controls. Duplication of the TBK1 gene is a rare cause of NTG. The identification of another case of NTG attributed to TBK1 gene duplication strengthens the case that this mutation causes glaucoma.

Molecular characterizations of grass carp (Ctenopharyngodon idella) TBK1 gene and its roles in regulating IFN-I pathway

TANK-binding kinase 1 (TBK1), a kinase at the crossroads of multiple IFN-inducing signaling pathways, plays essential roles in both antiviral and antibacterial innate immunity in mammals. Here, TBK1 gene (10339bp) was identified and characterized from grass carp Ctenopharyngodon idella (CiTBK1). The genomic sequence is shorter than other orthologs in vertebrate, and a promoter region is found in intron 1. mRNA expression of CiTBK1 was widespread in fifteen tissues investigated, and was up-regulated post GCRV challenge in vivo and in vitro, as well as after stimulation of viral/bacterial PAMPs in vitro. CiTBK1 mediates IFN-I signal pathway through over-expression experiment. Post GCRV challenge, CiTBK1 over-expression inhibits viral infection by induction of CiIFN-I and CiMx1 mainly via CiIRF7. In CiTBK1 over-expression cells, mRNA expressions of CiIRF3, CiIRF7 and CiIFN-I were inhibited, whereas CiMx1 was facilitated after poly I:C stimulation, comparing to those in control group. The result indicated that CiMx1 expression mediated by CiTBK1 is in IFN-I independent way after poly I:C stimulation. However, over-expression of CiTBK1 diminishes LPS-induced expressions of CiIRF3 and CiIRF7 but promotes the induction of CiIFN-I and CiMx1 in comparison with the control, which suggests that CiTBK1-triggered IFN-I activation is in IRF3/IRF7-independent manner after LPS stimulation. Notably, over-expression of CiTBK1 negatively regulated PGN-induced IRF3, IRF7, IFN-I and Mx1 immune response. Taken together, CiTBK1 participates in broad antiviral and antibacterial immune responses in different manners, and keeps regulatory balance that prevents harmful effects from excessive activation.

Copy number variations on chromosome 12q14 in patients with normal tension glaucoma

We report identification of a novel genetic locus (GLC1P) for normal tension glaucoma (NTG) on chromosome 12q14 using linkage studies of an African-American pedigree (maximum non-parametric linkage score = 19.7, max LOD score = 2.7). Subsequent comparative genomic hybridization and quantitative polymerase chain reaction (PCR) experiments identified a 780 kbp duplication within the GLC1P locus that is co-inherited with NTG in the pedigree. Real-time PCR studies showed that the genes within this duplication [TBK1 (TANK-binding kinase 1), XPOT, RASSF3 and GNS] are all expressed in the human retina. Cohorts of 478 glaucoma patients (including 152 NTG patients), 100 normal control subjects and 400 age-related macular degeneration patients were subsequently tested for copy number variation in GLC1P. Overlapping duplications were detected in 2 (1.3%) of the 152 NTG subjects, one of which had a strong family history of glaucoma. These duplications defined a 300 kbp critical region of GLC1P that spans two genes (TBK1 and XPOT). Microarray expression experiments and northern blot analysis using RNA obtained from human skin fibroblast cells showed that duplication of chromosome 12q14 results in increased TBK1 and GNS transcription. Finally, immunohistochemistry studies showed that TBK1 is expressed in the ganglion cells, nerve fiber layer and microvasculature of the human retina. Together, these data link the duplication of genes on chromosome 12q14 with familial NTG and suggest that an extra copy of the encompassed TBK1 gene is likely responsible for these cases of glaucoma. However, animal studies will be necessary to rule out a role for the other duplicated or neighboring genes.