The role of microtubule-associated protein tau in netrin-1 attractive signaling.

Direct binding of netrin receptors with dynamic microtubules (MTs) in the neuronal growth cone plays an important role in netrin-mediated axon guidance. However, how netrin-1 (NTN1) regulates MT dynamics in axon turning remains a major unanswered question. Here, we show that the coupling of netrin-1 receptor DCC with tau (MAPT)-regulated MTs is involved in netrin-1-promoted axon attraction. Tau directly interacts with DCC and partially overlaps with DCC in the growth cone of primary neurons. Netrin-1 induces this interaction and the colocalization of DCC and tau in the growth cone. The netrin-1-induced interaction of tau with DCC relies on MT dynamics and TUBB3, a highly dynamic ß-tubulin isotype in developing neurons. Netrin-1 increased cosedimentation of DCC with tau and TUBB3 in MTs, and knockdown of either tau or TUBB3 mutually blocked this effect. Downregulation of endogenous tau levels by tau shRNAs inhibited netrin-1-induced axon outgrowth, branching and commissural axon attraction in vitro, and led to defects in spinal commissural axon projection in vivo. These findings suggest that tau is a key MT-associated protein coupling DCC with MT dynamics in netrin-1-promoted axon attraction.

Lactate promotes neuronal differentiation of SH-SY5Y cells by lactate-responsive gene sets through NDRG3-dependent and -independent manners.

Lactate serves as the major glucose alternative to an energy substrate in the brain. Lactate level is increased in the fetal brain from the middle stage of gestation, indicating the involvement of lactate in brain development and neuronal differentiation. Recent reports show that lactate functions as a signaling molecule to regulate gene expression and protein stability. However, the roles of lactate signaling in neuronal cells remain unknown. Here, we showed that lactate promotes the all stages of neuronal differentiation of SH-SY5Y and Neuro2A, human and mouse neuroblastoma cell lines, characterized by increased neuronal marker expression and the rates of neurites extension. Transcriptomics revealed many lactate-responsive genes sets such as SPARCL1 in SH-SY5Y, Neuro2A, and primary embryonic mouse neuronal cells. The effects of lactate on neuronal function were mainly mediated through monocarboxylate transporters 1 (MCT1). We found that NDRG family member 3 (NDRG3), a lactate-binding protein, was highly expressed and stabilized by lactate treatment during neuronal differentiation. Combinative RNA-seq of SH-SY5Y with lactate treatment and NDRG3 knockdown shows that the promotive effects of lactate on neural differentiation are regulated through NDRG3-dependent and independent manners. Moreover, we identified TEA domain family member 1 (TEAD1) and ETS-related transcription factor 4 (ELF4) are the specific transcription factors that are regulated by both lactate and NDRG3 in neuronal differentiation. TEAD1 and ELF4 differently affect the expression of neuronal marker genes in SH-SY5Y cells. These results highlight the biological roles of extracellular and intracellular lactate as a critical signaling molecule that modifies neuronal differentiation.

NOTCH3 promotes docetaxel resistance of prostate cancer cells through regulating TUBB3 and MAPK signaling pathway.

Docetaxel is the preferred chemotherapeutic agent in patients with castrate-resistant prostate cancer (CRPC). However, patients eventually develop docetaxel resistance and in the absence of effective treatment options. Consequently, it is essential to investigate the mechanisms generating docetaxel resistance and develop novel alternative therapeutic targets. RNA sequencing was undertaken on docetaxel-sensitive and docetaxel-resistant prostate cancer (PCa) cells. Subsequently, chemoresistance, cancer stemness, and lipid metabolism were investigated. To obtain insight into the precise activities and action mechanisms of NOTCH3 in docetaxel-resistant PCa, immunoprecipitation, mass spectrometry, ChIP, luciferase reporter assay, cell metabolism, and animal experiments were performed. Through RNA sequencing analysis, we found that NOTCH3 expression was markedly higher in docetaxel-resistant cells relative to parental cells, and that this trend was continued in docetaxel-resistant PCa tissues. Experiments in vitro and in vivo revealed that NOTCH3 enhanced stemness, lipid metabolism, and docetaxel resistance in PCa. Mechanistically, NOTCH3 is bound to TUBB3 and activates the MAPK signaling pathway. Moreover, NOTCH3 was directly regulated by MEF2A in docetaxel-resistant cells. Notably, targeting NOTCH3 and the MEF2A/TUBB3 signaling axis was related to docetaxel chemoresistance in PCa. Overall, these results demonstrated that NOTCH3 fostered stemness, lipid metabolism, and docetaxel resistance in PCa via the TUBB3 and MAPK signaling pathways. Therefore, NOTCH3 may be employed as a prognostic biomarker in PCa patients. NOTCH3 could be a therapeutic target for PCa patients, particularly those who have developed docetaxel resistance.

Axonal precursor miRNAs hitchhike on endosomes and locally regulate the development of neural circuits.

Various species of non-coding RNAs (ncRNAs) are enriched in specific subcellular compartments, but the mechanisms orchestrating their localization and their local functions remain largely unknown. We investigated both aspects using the elongating retinal ganglion cell axon and its tip, the growth cone, as models. We reveal that specific endogenous precursor microRNAs (pre-miRNAs) are actively trafficked to distal axons by hitchhiking primarily on late endosomes/lysosomes. Upon exposure to the axon guidance cue semaphorin 3A (Sema3A), pre-miRNAs are processed specifically within axons into newly generated miRNAs, one of which, in turn, silences the basal translation of tubulin beta 3 class III (TUBB3), but not amyloid beta precursor protein (APP). At the organismal level, these mature miRNAs are required for growth cone steering and a fully functional visual system. Overall, our results uncover a novel mode of ncRNA transport from one cytosolic compartment to another within polarized cells. They also reveal that newly generated miRNAs are critical components of a ncRNA-based signaling pathway that transduces environmental signals into the structural remodeling of subcellular compartments.

The prognostic and predictive role of class III ß-Tubulin and hENT1 expression in patients with advanced pancreatic ductal adenocarcinoma.

BACKGROUND: FOLFIRINOX and gemcitabine-nabpaclitaxel (GnP) are standard first-line treatment regimens for advanced pancreatic ductal adenocarcinoma (PDAC). However, currently, there is a lack of predictive biomarkers to aid in the treatment selection. We aimed to explore the prognostic and predictive value of class III ß-Tubulin (TUBB3) and human equilibrative nucleoside transporter 1 (hENT1) expression, which have previously been shown to be associated with taxane and gemcitabine resistance in advanced PDAC. METHODS: We conducted a retrospective analysis of 106 patients with advanced PDAC treated with GnP and/or FOLFIRINOX at our institution. TUBB3 and hENT1 immunohistochemical staining was performed on tumor specimens and subsequently evaluated based on the intensity and percentage of expression. RESULTS: In patients who received the GnP regimen, a high combined score (TUBB3low/hENT1high) was associated with a higher DCR and longer PFS compared to those with intermediate (TUBB3high/hENT1high or TUBB3low/hENT1low) and low score (TUBB3high/hENT1low). In the multivariate analysis, a high combined score was an independent predictor of higher DCR (OR:11.96; 95 % CI:2.61-54.82; p = 0.001) and longer PFS (HR:0.33; 95%CI:0.18-0.60; p < 0.001). However, there was no difference in response rates or PFS based on TUBB3 and hENT1 expression among patients receiving the FOLFIRINOX regimen. CONCLUSION: Our findings indicate that tumor TUBB3 and hENT1 expression may predict the efficacy of the GnP regimen, and low TUBB3 and high hENT1 expression (TUBB3low/hENT1high) are associated with a higher DCR and longer PFS in patients treated with GnP. Evaluating TUBB3 and hENT1 jointly can identify the patients most (as well as least) likely to benefit from GnP chemotherapy.

TUBB4B is a novel therapeutic target in non-alcoholic fatty liver disease-associated hepatocellular carcinoma.

Non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC) is an emerging malignancy due to the rising prevalence of NAFLD. However, no drug is available to target NAFLD-HCC. In this study, we aim to unravel novel therapeutic targets of NAFLD-HCC utilizing a high-throughput CRISPR/Cas9 screening strategy. We utilized the Epi-drug CRISPR/Cas9 library consisting of single-guide RNAs (sgRNAs) targeting over 1,000 genes representing the FDA-approved drug targets and epigenetic regulators to perform loss-of-function screening in two NAFLD-HCC cell lines (HKCI2 and HKCI10). CRISPR/Cas9 library screening unraveled TUBB4B as an essential gene for NAFLD-HCC cell growth. TUBB4B was overexpressed in NAFLD-HCC tumors compared with adjacent normal tissues (N = 17) and was associated with poor survival (p < 0.01). RNA-sequencing and functional assays revealed that TUBB4B knockout in NAFLD-HCC promoted cell apoptosis, cell cycle arrest, and cellular senescence, leading to suppressed NAFLD-HCC growth in vitro and in vivo. We identified that TUBB4B inhibitor mebendazole (MBZ), an FDA-approved drug, inhibited NAFLD-HCC growth by inducing apoptosis and cellular senescence. Since protein expression of pro-survival Bcl-xL was induced in TUBB4B knockout NAFLD-HCC cells, we examined combination of TUBB4B inhibition with navitoclax, a Bcl-xL inhibitor that selectively targets senescent cells. Consistent with our hypothesis, either TUBB4B knockout or MBZ synergized with navitoclax to inhibit NAFLD-HCC cell growth via the induction of intrinsic and extrinsic apoptosis pathways. In summary, TUBB4B is a novel therapeutic target in NAFLD-HCC. Inhibition of TUBB4B with MBZ in combination with navitoclax synergistically inhibited NAFLD-HCC cell growth, representing a promising strategy for the treatment of NAFLD-HCC. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Identification and Validation of Prognostic Markers for Endometriosis-Associated Ovarian Cancer.

Background: Growing evidence suggests that endometriosis (EMs) is a risk factor for endometriosis-associated ovarian cancer (EAOC). The aim was to identify and validate gene signatures associated with EMs that may serve as potential biomarkers for evaluating the prognosis of patients with EAOC. Methods: The data of EMs and control samples was obtained from GEO database. The weighted gene co-expression network analysis (WGCNA) identified modular genes significantly associated with EMs. The KEGG pathway and GO functional enrichment analyses were also performed. Univariate Cox regression analysis was conducted to screen marker genes associated with the prognosis of EAOC patients. Finally, RT-qPCR and immunohistochemical verified the expression of ADAMTS19 and TUBB in normal ovarian and EAOC tissues, and the biological functions of ADAMTS19 and TUBB were preliminarily explored by CCK8 and Transwell assays. Results: The WGCNA identified 2 co-expression modules, which in total included 615 genes, and 7642 differentially expressed genes (DEGs) were detected thorough analysis of the EAOC dataset. After taking the intersection of 615 modular genes and 7642 DEGs, 214 shared genes were obtained, and univariate COX regression analysis pointed 10 genes associated with the prognosis of EAOC. Moreover, it was demonstrated by RT-qPCR and immunohistochemical staining experiments that ADAMTS19 expression was elevated, while TUBB expression was reduced in EAOC compared with normal ovarian cells and tissues. Finally, cell experiments revealed that ADAMTS19 promoted the proliferation and invasion in EAOC cells, while overexpression of TUBB inhibited these processes. Conclusions: The present study identified and validated new EMs-associated gene markers, which could serve as potential biomarkers for assessing the prognostic risk of EAOC patients. In addition, some of these genes may have significance as novel therapeutic targets and could be used to guide clinical applications.

OSGIN1 is a novel TUBB3 regulator that promotes tumor progression and gefitinib resistance in non-small cell lung cancer.

BACKGROUND: Oxidative stress induced growth inhibitor 1 (OSGIN1) regulates cell death. The role and underlying molecular mechanism of OSGIN1 in non-small cell lung cancer (NSCLC) are uncharacterized. METHODS: OSGIN1 expression in NSCLC samples was detected using immunohistochemistry and Western blotting. Growth of NSCLC cells and gefitinib-resistant cells expressing OSGIN1 or TUBB3 knockdown was determined by MTT, soft agar, and foci formation assays. The effect of OSGIN1 knockdown on in vivo tumor growth was assessed using NSCLC patient-derived xenograft models and gefitinib-resistant patient-derived xenograft models. Potentially interacting protein partners of OSGIN1 were identified using IP-MS/MS, immunoprecipitation, PLA, and Western blotting assays. Microtubule dynamics were explored by tubulin polymerization assay and immunofluorescence. Differential expression of signaling molecules in OSGIN1 knockdown cells was investigated using phospho-proteomics, KEGG analysis, and Western blotting. RESULTS: We found that OSGIN1 is highly expressed in NSCLC tissues and is positively correlated with low survival rates and tumor size in lung cancer patients. OSGIN1 knockdown inhibited NSCLC cell growth and patient-derived NSCLC tumor growth in vivo. Knockdown of OSGIN1 strongly increased tubulin polymerization and re-established gefitinib sensitivity in vitro and in vivo. Additionally, knockdown of TUBB3 strongly inhibited NSCLC cell proliferation. Mechanistically, we found that OSGIN1 enhances DYRK1A-mediated TUBB3 phosphorylation, which is critical for inducing tubulin depolymerization. The results of phospho-proteomics and ontology analysis indicated that knockdown of OSGIN1 led to reduced propagation of the MKK3/6-p38 signaling axis. CONCLUSIONS: We propose that OSGIN1 modulates microtubule dynamics by enhancing DYRK1A-mediated phosphorylation of TUBB3 at serine 172. Moreover, elevated OSGIN1 expression promotes NSCLC tumor growth and gefitinib resistance through the MKK3/6-p38 signaling pathway. Our findings unveil a new mechanism of OSGIN1 and provide a promising therapeutic target for NSCLC treatment in the clinic.

Quantitative proteomics identifies TUBB6 as a biomarker of muscle-invasion and poor prognosis in bladder cancer.

Muscle-invasive urothelial carcinoma (MIUC) of the bladder shows highly aggressive tumor behavior, which has prompted the quest for robust biomarkers predicting invasion. To discover such biomarkers, we first employed high-throughput proteomic method and analyzed tissue biopsy cohorts from patients with bladder urothelial carcinoma (BUC), stratifying them according to their pT stage. Candidate biomarkers were selected through bioinformatic analysis, followed by validation. The latter comprised 2D and 3D invasion and migration assays, also a selection of external public datasets to evaluate mRNA expression and an in-house patient-derived tissue microarray (TMA) cohort to evaluate protein expression with immunohistochemistry (IHC). Our multilayered platform-based analysis identified tubulin beta 6 class V (TUBB6) as a promising prognostic biomarker predicting MIUC of the bladder. The in vitro 2D and 3D migration and invasion assays consistently showed that inhibition of TUBB6 mRNA significantly reduced cell migration and invasion ability in two BUC cell lines with aggressive phenotype (TUBB6 migration, P = .0509 and P < .0001; invasion, P = .0002 and P = .0044; TGFBI migration, P = .0214 and P = .0026; invasion, P < .0001 and P = .0001; T24 and J82, respectively). Validation through multiple public datasets, including The Cancer Genome Atlas (TCGA) and selected GSE (Genomic Spatial Event) databases, confirmed TUBB6 as a potential biomarker predicting MIUC. Further protein-based validation with our TMA cohort revealed concordant results, highlighting the clinical implication of TUBB6 expression in BUC patients (overall survival: P < .001). We propose TUBB6 as a novel IHC biomarker to predict invasion and poor prognosis, also select the optimal treatment in BUC patients.

Inherited bone marrow failure with macrothrombocytopenia due to germline tubulin beta class I (TUBB) variant.

Germline mutations in tubulin beta class I (TUBB), which encodes one of the ß-tubulin isoforms, were previously associated with neurological and cutaneous abnormalities. Here, we describe the first case of inherited bone marrow (BM) failure, including marked thrombocytopenia, morphological abnormalities, and cortical dysplasia, associated with a de novo p.D249V variant in TUBB. Mutant TUBB had abnormal cellular localisation in transfected cells. Following interferon/ribavirin therapy administered for transfusion-acquired hepatitis C, severe pancytopenia and BM aplasia ensued, which was unresponsive to immunosuppression. Acquired chromosome arm 6p loss of heterozygosity was identified, leading to somatic loss of the mutant TUBB allele.

Nuclear-cytoplasmic asynchrony in oocyte maturation caused by TUBB8 variants via impairing microtubule function: a novel pathogenic mechanism.

BACKGROUND: TUBB8, a crucial gene encoding microtubule protein, plays a pivotal role in cellular processes. Deleterious TUBB8 variants have been shown to significantly hinder oocyte maturation. In this study, we conducted an in vitro investigation using TUBB8 mutant mouse oocytes to elucidate the pathogenic mechanisms of TUBB8 variants in oocyte nuclear and cytoplasmic maturation. METHODS: A mutant model was successfully established in mouse oocytes via microinjection to further investigate the effects of four novel discovered TUBB8 mutations on the nuclear and cytoplasmic maturation of mouse oocytes. Immunofluorescence and confocal microscopy were performed to observe the cortical polarity and spindle and of mutant oocytes. Active mitochondrial staining was performed to analyze mitochondrial distribution patterns. Endoplasmic reticulum and Ca2+ staining were conducted to assess ER distribution and cytoplasmic calcium ion concentration in oocytes. RESULTS: In mouse oocytes, TUBB8 variants (p.A313V, p.C239W, p.R251Q, and p.G96R) resulted in a reduction of the first polar body extrusion rate, disruption of spindle assembly, and abnormal chromosome distribution. Additionally, these variants induced oocyte organelle abnormalities, including anomalies in mitochondrial redistribution and endoplasmic reticulum stress compared to the wild-type. CONCLUSION: Deleterious TUBB8 variants could disrupt microtubule function, affecting critical processes such as spindle assembly, chromosome distribution, and organelle rearrangement during oocyte meiosis. These disruptions culminate in compromised nuclear-cytoplasmic maturation, consequently giving rise to oocyte maturation defects.

Genetic screening and analysis of TUBB8 variants in females seeking ART.

RESEARCH QUESTION: More than 100 variants have been identified in the TUBB8 gene, which account for approximately 30% of infertile women with oocyte maturation defects. But what is the correlation between the highly phenotypic diversity and genetic variability? Are there other variants in TUBB8 related to female infertility? DESIGN: TUBB8 resequencing was performed in 80 female subjects who were experiencing infertility and were seeking treatment with assisted reproductive technologies (ART), or had ever experienced ART failure due to oocyte maturation defects. All variants were evaluated with pedigree analysis, population frequency, in-silico analysis and molecular modelling. The effects of the variants on oocytes/arrested embryos were assessed by morphological observations, immunostaining, embryo biopsies and chromosome euploidy analysis. RESULTS: Nine missense variants and two frameshift variants from an additional 15 families were identified, including four novel variants and seven previously reported recurrent variants. These TUBB8 variants were related to highly variable phenotypes, including abnormalities in oocyte maturation or morphology, fertilization failure, embryonic development abnormalities and implantation failure. Also further clarified were the incomplete penetrance of heterozygous p.E108K, the likely benign significance of heterozygous p.A313V and the clinical effect of a novel variant of p.R380C. CONCLUSIONS: This study significantly expands the variant spectrum of the TUBB8 gene and, together with the available findings on TUBB8 variants and female infertility, will potentially facilitate the genetic counselling of infertile women in future.

Evaluation of TUBB8 gene alterations in infertile women with oocyte maturation and cleavage arrest referred to Royan Institute.

RESEARCH QUESTION: Are TUBB8 gene variations present in Iranian infertile women with oocyte maturation arrest or embryo cleavage arrest? DESIGN: TUBB8 gene variations were investigated by polymerase chain reaction sequencing on blood samples from 16 women with oocyte maturation arrest and 12 women with cleavage arrest, collectively referred to as the experimental cohort, as well as 56 fertile women as the control group. The Exome Sequencing Project and dbSNP databases and the Genome Aggregation Database were used to search the frequency of corresponding variants. PolyPhen and SIFT were used to conduct in-silico analysis of gene variations and Align-GVGD was used to predict the effect of missense variants on proteins. The homology modelling and structure evaluation of variations was also checked. RESULTS: Two likely pathogenic variants [c.713C>T (p.Thr238Met), c.1054G>T (p.Ala352Ser)] were identified in patients with oocyte maturation arrest and one likely pathogenic variant [c.G763A, (p.Val255Met)] was identified in a patient with cleavage arrest. These changes were absent in controls. CONCLUSIONS: Three deleterious variants in TUBB8 related to oocyte maturation arrest or cleavage arrest and infertility were identified. TUBB8 variant screening for patients with oocyte maturation and cleavage arrest is recommended.

Clinicopathological significance of TUBB3 in upper tract urothelial carcinoma and possible application in urine cytology.

ßIII-Tubulin, encoded by the TUBB3 gene, is a microtubule protein. We previously reported that TUBB3 is overexpressed in renal cell carcinoma. We investigated the clinicopathological significance of TUBB3 in upper tract urothelial carcinoma (UTUC) by immunohistochemistry. In normal tissue, TUBB3 expression was weak or absent. In contrast, TUBB3 overexpression was observed in urothelial carcinoma (UC) tissues in 51 (49%) of 103 UTUC cases. TUBB3 overexpression was associated with nodular/flat morphology, high-grade disease, high T stage, and a poor prognosis. Similar results were obtained in The Cancer Genome Atlas bladder cancer cohort. TUBB3 expression was also associated with high Ki-67 labeling index, CD44v9, HER2, EGFR, and p53 expression in UTUC. Among representative cancer-related molecules, TUBB3 was an independent predictor of progression-free survival and high-grade UC. Finally, using urine cytology samples, we analyzed TUBB3 expression by immunocytochemistry. TUBB3 expression was more frequently found in UC cells than in nonneoplastic cells. The diagnostic accuracy of urine cytology was improved when combined with TUBB3 immunostaining. The findings suggest the importance of TUBB3 in tumor progression and its potential application as a biomarker for high-grade disease and the prognosis of UC. Moreover, combination with TUBB3 immunostaining might improve the diagnostic accuracy of urine cytology.

Tubb3 expression levels are sensitive to neuronal activity changes and determine microtubule growth and kinesin-mediated transport.

Microtubules are dynamic polymers of α/ß-tubulin. They regulate cell structure, cell division, cell migration, and intracellular transport. However, functional contributions of individual tubulin isotypes are incompletely understood. The neuron-specific ß-tubulin Tubb3 displays highest expression around early postnatal periods characterized by exuberant synaptogenesis. Although Tubb3 mutations are associated with neuronal disease, including abnormal inhibitory transmission and seizure activity in patients, molecular consequences of altered Tubb3 levels are largely unknown. Likewise, it is unclear whether neuronal activity triggers Tubb3 expression changes in neurons. In this study, we initially asked whether chemical protocols to induce long-term potentiation (cLTP) affect microtubule growth and the expression of individual tubulin isotypes. We found that growing microtubules and Tubb3 expression are sensitive to changes in neuronal activity and asked for consequences of Tubb3 downregulation in neurons. Our data revealed that reduced Tubb3 levels accelerated microtubule growth in axons and dendrites. Remarkably, Tubb3 knockdown induced a specific upregulation of Tubb4 gene expression, without changing other tubulin isotypes. We further found that Tubb3 downregulation reduces tubulin polyglutamylation, increases KIF5C motility and boosts the transport of its synaptic cargo N-Cadherin, which is known to regulate synaptogenesis and long-term potentiation. Due to the large number of tubulin isotypes, we developed and applied a computational model based on a Monte Carlo simulation to understand consequences of tubulin expression changes in silico. Together, our data suggest a feedback mechanism with neuronal activity regulating tubulin expression and consequently microtubule dynamics underlying the delivery of synaptic cargoes.

A novel compound heterozygous mutation in TUBB8 causing early embryonic developmental arrest.

PURPOSE: Mutations in the ß-tubulin isotype, TUBB8, can cause female infertility. Although several mutations of TUBB8 have been reported, the full spectrum for guiding genetics counseling still needs to be further explored. Here, we sought to identify novel variants in TUBB8 and their phenotypic effects on microtubule network structure in vitro. METHODS: Whole-exome sequence analysis was performed in two families with infertility to detect pathogenic variants, with validation by Sanger sequencing. All gene variants and protein structures were predicted in silico. Cells were transfected with wild-type and mutants, and immunofluorescence analysis was performed to visualize microtubule network changes. RESULTS: We detected a novel compound heterozygous mutation, c.915_916delCC (p.Arg306Serfs*21) and c.82C > T (p.His28Tyr), and a benign heterozygous variant c.1286C > T (p.Thr429Met) in TUBB8 in the two families. Female patients with p.Arg306Serfs*21 and p.His28Tyr were infertile with early embryonic developmental arrest. The female patient with p.Thr429Met gave birth to a healthy baby in the second in vitro fertilization frozen embryo transfer cycle. The p.Arg306Serfs*21 mutation was predicted to cause large structural alteration in the TUBB8 protein and was confirmed to produce a truncated and trace protein by western blot analysis. Immunofluorescence analysis of transfected HeLa cells showed that p.Arg306Serfs*21 significantly disrupted microtubule structure. CONCLUSIONS: Our findings expand the known mutational spectrum of TUBB8 associated with early embryonic developmental arrest and female infertility.

Primary myeloid cell proteomics and transcriptomics: importance of ß-tubulin isotypes for osteoclast function.

Among hematopoietic cells, osteoclasts (OCs) and immature dendritic cells (DCs) are closely related myeloid cells with distinct functions: OCs participate skeleton maintenance while DCs sample the environment for foreign antigens. Such specificities rely on profound modifications of gene and protein expression during OC and DC differentiation. We provide global proteomic and transcriptomic analyses of primary mouse OCs and DCs, based on original stable isotope labeling with amino acids in cell culture (SILAC) and RNAseq data. We established specific signatures for OCs and DCs, including genes and proteins of unknown functions. In particular, we showed that OCs and DCs have the same α- and ß-tubulin isotype repertoire but that OCs express much more of the ß tubulin isotype Tubb6 (also known as TBB6). In both mouse and human OCs, we demonstrate that elevated expression of Tubb6 in OCs is necessary for correct podosomes organization and thus for the structure of the sealing zone, which sustains the bone resorption apparatus. Hence, lowering Tubb6 expression hinders OC resorption activity. Overall, we highlight here potential new regulators of OC and DC biology, and illustrate the functional importance of the tubulin isotype repertoire in the biology of differentiated cells.

TUBB3 immunostaining improves the diagnostic accuracy of oral liquid-based cytology in squamous cell carcinoma.

OBJECTIVE: Although class III beta-tubulin (TUBB3) is not expressed in normal epithelium, its expression in cancers of some organs has been reported. Herein, we investigated the expression pattern and expression levels of TUBB3 in oral squamous cell carcinoma (SCC), and assessed whether TUBB3 immunostaining could improve the diagnostic accuracy of oral scraping liquid-based cytology (LBC). METHODS: Paraffin sections of biopsies from 107 patients with primary SCC and 30 patients with squamous papilloma of the tongue or gingiva were immunostained for TUBB3. In addition, 15 LBC samples obtained from the study participants with SCC were immunostained for TUBB3. Seven LBC samples were false-negative. The TUBB3 expression level in each sample was evaluated and classified as 3+, 2+, 1+, or 0. RESULTS: TUBB3 expression was confirmed in 91.6% of paraffin-embedded SCC specimens. Clear and diffuse positivity (2+ or above) was observed in 77.6% of the total cases. In the well-differentiated type, tumour cells in the middle layer of the parenchyma specifically expressed TUBB3. In almost LBC samples, cancerous intermediate cells showed immunopositivity similar to that of paraffin samples, even if cellular atypia was not clear in Papanicolaou staining. CONCLUSIONS: TUBB3 immunostaining is useful for diagnosing oral SCC in scraping LBC, especially when samples consist of intermediate cells with little morphological change. Moreover, TUBB3 immunostaining could improve the diagnostic accuracy of oral scraping LBC by reducing false-negatives.

Maternal Germline Mosaicism of a de Novo TUBB2B Mutation Leads to Complex Cortical Dysplasia in Two Siblings.

Introduction: Complex cortical dysplasia with other brain malformations-7 (a.k.a. polymicrogyria) caused by mutations in TUBB2B gene is a clinically heterogeneous condition. Case report: We report two siblings with polymicrogyria. Brain MRI showed polymicrogyria, small brainstem, thin corpus callosum and fused basal ganglia. Karyotypes and chromosomal microarray analysis were normal. By whole exome sequencing, there were a de novo variant of c.728C > T (p.P243L) in both siblings and a common single nucleotide polymorphism (SNP) (c.718C > T) in both siblings and the mother. Seminal DNA analysis obtained from father was normal. Conclusion: Maternal germline mosaicism was considered because the sequencing result of the father's sperm was normal, two siblings had the same disease, and both patients and mother had the same SNP.

Cross-sectional quantitative analysis of the natural history of TUBA1A and TUBB2B tubulinopathies.

PURPOSE: TUBA1A and TUBB2B tubulinopathies are rare neurodevelopmental disorders characterized by cortical and extracortical malformations and heterogenic phenotypes. There is a need for quantitative clinical endpoints that will be beneficial for future diagnostic and therapeutic trials. METHODS: Quantitative natural history modeling of individuals with TUBA1A and TUBB2B tubulinopathies from clinical reports and database entries of DECIPHER and ClinVar. Main outcome measures were age at disease onset, survival, and diagnostic delay. Phenotypical, neuroradiological, and histopathological features were descriptively illustrated. RESULTS: Mean age at disease onset was 4 (TUBA1A) and 6 months (TUBB2B), respectively. Mortality was equally estimated with 7% at 3.2 (TUBA1A) and 8.0 years (TUBB2B). Diagnostic delay was significantly higher in TUBB2B (12.3 years) compared with TUBA1A tubulinopathy (4.2 years). We delineated the isotype-dependent clinical, neuroradiological, and histopathological phenotype of affected individuals and present brain malformations associated with epilepsy and an unfavorable course of disease. CONCLUSION: The natural history of tubulinopathies is defined by the genotype and associated brain malformations. Defined data on estimated survival, diagnostic delay, and disease characteristics of TUBA1A and TUBB2B tubulinopathy will help to raise disease awareness and encourage future clinical trials to optimize genetic testing, family counseling, and supportive care.