Tubulin Polymerization-promoting Protein Research Articles

TPPP-BRD9 fusion-related gallbladder carcinomas are frequently associated with intracholecystic neoplasia, neuroendocrine carcinoma, and a distinctive small tubular-type adenocarcinoma commonly accompanied with a syringomatous pattern

A fusion between tubulin polymerization-promoting protein (TPPP), a regulatory cytoskeletal gene, and the chromatin remodeling factor, bromodomain-containing protein 9 (BRD9), TPPP-BRD9 fusion has been found in rare cancer cases, including lung and gallbladder cancers (GBC). In this study, we investigated the histopathological features of 16 GBCs previously shown by RNA sequencing to harbor the TPPP-BRD9 fusion. Findings in the fusion-positive GBCs were compared with 645 GBC cases from the authors’ database. Among the 16 TPPP-BRD9 fusion-positive GBC cases, most were females (F:M = 7:1) of Chinese ethnicity (12/16), whereas the remaining cases were from Chile. The histopathological examination showed the following findings: 1) Intracholecystic neoplasm (ICN) in 7/15 (47% vs. 7% 645 reference GBCs, p < 0.001), all with gastro-pancreatobiliary phenotype, often with clear cell change, and in the background of pyloric gland metaplasia and extensive high-grade dysplasia. 2) Neuroendocrine carcinoma (NEC) morphology: 3 cases (27% vs. 4.6% in the reference database, p = 0.001) showed a sheet-like and nested/trabecular growth pattern of monotonous cells with salt-and-pepper chromatin characteristic of NECs. Two were large cell type, one had prominent clear cell features, a rare finding in GBNECs; the other one had relatively bland, well-differentiated morphology, and the remaining case was small cell type. 3) Adenocarcinoma identified in 8 cases had a distinctive pattern characterized by widely separated small, round tubular units with relatively uniform nuclei in a fashion seen in mesonephric adenocarcinomas, including hobnail-like arrangement and apical snouts, reminiscent of tubular carcinomas of the breast in many areas. In some foci, the epithelium was attenuated, and glands were elongated, some with comma shapes, which along with the mucinous/necrotic intraluminal debris created a “syringoid” appearance. 4) Other occasional patterns included the cribriform, glomeruloid patterns, and metaplastic tubular-spindle cell pattern accompanied by hemorrhage. In conclusion, TPPP-BRD9 fusion-positive GBCs often develop through intracholecystic neoplasms (adenoma-carcinoma sequence) of gastro-pancreatobiliary lineage, appear more prone to form NEC morphology and have a propensity to display clear cell change. Invasive adenocarcinomas arising in this setting often seem to display a distinctive appearance that we tentatively propose as the TPPP-BRD9 fusion-positive pattern of GBC.

SNCA and TPPP transcripts increase in oligodendroglial cytoplasmic inclusions in multiple system atrophy

Multiple system atrophy (MSA) is characterized by glial cytoplasmic inclusions (GCIs) containing aggregated α-synuclein (α-syn) in oligodendrocytes. The origin of α-syn accumulation in GCIs is unclear, in particular whether abnormal α-syn aggregates result from the abnormal elevation of endogenous α-syn expression in MSA or ingested from the neuronal source. Tubulin polymerization promoting protein (TPPP) has been reported to play a crucial role in developing GCI pathology. Here, the total cell body, nucleus, and cytoplasmic area density of SNCA and TPPP transcripts in neurons and oligodendrocytes with and without various α-syn pathologies in the pontine base in autopsy cases of MSA (n = 4) and controls (n = 2) were evaluated using RNAscope with immunofluorescence. Single-nucleus RNA-sequencing data for TPPP was evaluated using control frontal cortex (n = 3). SNCA and TPPP transcripts were present in the nucleus and cytoplasm of oligodendrocytes in both controls and diseased, with higher area density in GCIs and glial nuclear inclusions in MSA. Area densities of SNCA and TPPP transcripts were lower in neurons showing cytoplasmic inclusions in MSA. Indeed, TPPP transcripts were unexpectedly found in neurons, while the anti-TPPP antibody failed to detect immunoreactivity. Single-nucleus RNA-sequencing revealed significant TPPP transcript expression predominantly in oligodendrocytes, but also in excitatory and inhibitory neurons. This study addressed the unclear origin of accumulated α-syn in GCIs, proposing that the elevation of SNCA transcripts may supply templates for misfolded α-syn. In addition, the parallel behavior of TPPP and SNCA transcripts in GCI development highlights their potential synergistic contribution to inclusion formation. In conclusion, this study advances our understanding of MSA pathogenesis, offers insights into the dynamics of SNCA and TPPP transcripts in inclusion formation, and proposes regulating their transcripts for future molecular therapy to MSA.

PERITENON AND SYNOVIAL TPPP3+ PROGENITOR CELLS CONTRIBUTE TO POST-TRAUMATIC HETEROTOPIC BONE FORMATION

Heterotopic ossification (HO) is defined as aberrant bone formation in extraskeletal locations. In this process, local stromal cells of mesenchymal origin abnormally differentiate, resulting in pathologic cartilage and bone matrix deposition. However, the specific cell type and mechanisms beyond this process are not well understood, in part due to the heterogeneity of progenitor cells involved. Here, a combination of single cell RNA sequencing (scRNA-Seq) and lineage tracing, defined the extent to which synovial / tendon sheath progenitor cells contribute to HO. For this purpose, a Tppp3 (tubulin polymerization-promoting protein family member 3) inducible reporter model was used, in combination with either Scx (Scleraxis) or Pdgfra (Platelet derived growth factor receptor alpha) reporter animals. Both arthroplasty-induced and tendon injury-mouse experimental HO models were utilized. ScRNA-Seq of tendon-induced traumatic HO suggested that Tppp3 is a progenitor cell marker for either osteochondral or tendon or cells. After HO induction, Tppp3 reporter+ cell population expanded in number and contributed to cartilage and bone formation in tendon and joint-associated HO. Using double reporter animals, we found that both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells produced HO-associated cartilage. Finally, the examination of human samples showed a significant population of TPPP3+ cells overlapping with osteogenic markers in areas of HO. Overall, these results provide novel observations that peritenon and synovial progenitor cells undergo abnormal osteochondral differentiation and contribute to heterotopic bone formation after trauma.

The Unicellular, Parasitic Fungi, Sanchytriomycota, Possess a DNA Sequence Possibly Encoding a Long Tubulin Polymerization Promoting Protein (TPPP) but Not a Fungal-Type One.

The unicellular, parasitic fungi of the phylum Sanchytriomycota (sanchytrids) were discovered a few years ago. These unusual chytrid-like fungi parasitize algae. The zoospores of the species of the phylum contain an extremely long kinetosome composed of microtubular singlets or doublets and a non-motile pseudocilium (i.e., a reduced posterior flagellum). Fungi provide an ideal opportunity to test and confirm the correlation between the occurrence of flagellar proteins (the ciliome) and that of the eukaryotic cilium/flagellum since the flagellum occurs in the early-branching phyla and not in terrestrial fungi. Tubulin polymerization promoting protein (TPPP)-like proteins, which contain a p25alpha domain, were also suggested to belong to the ciliome and are present in flagellated fungi. Although sanchytrids have lost many of the flagellar proteins, here it is shown that they possess a DNA sequence possibly encoding long (animal-type) TPPP, but not the fungal-type one characteristic of chytrid fungi. Phylogenetic analysis of p25alpha domains placed sanchytrids into a sister position to Blastocladiomycota, similarly to species phylogeny, with maximal support.

Endometrial proteomic profile of patients with repeated implantation failure.

Successful embryo implantation, is the initiating step of pregnancy, relies on not only the high quality of the embryo but also the synergistic development of a healthy endometrium. Characterization and identification of biomarkers for the receptive endometrium is an effective method for increasing the probability of successful embryo implantation. Endometrial tissues from 22 women with a history of recurrent implantation failure (RIF) and 19 fertile controls were collected using biopsy catheters on 7-9 days after the peak of luteinizing hormone. Differentially expressed proteins (DEPs) were identified in six patients with RIF and six fertile controls using isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics analysis. Two hundred and sixty-three DEPs, including proteins with multiple bioactivities, such as protein translation, mitochondrial function, oxidoreductase activity, fatty acid and amino acid metabolism, were identified from iTRAQ. Four potential biomarkers for receptive endometrium named tubulin polymerization-promoting protein family member 3 TPPP3, S100 Calcium Binding Protein A13 (S100A13), 17b-hydroxysteroid dehydrogenase 2 (HSD17B2), and alpha-2-glycoprotein 1, zinc binding (AZGP1) were further verified using ProteinSimple Wes and immunohistochemical staining in all included samples (n=22 for RIF and n=19 for controls). Of the four proteins, the protein levels of TPPP3 and HSD17B2 were significantly downregulated in the endometrium of patients with RIF. Poor endometrial receptivity is considered the main reason for the decrease in pregnancy success rates in patients suffering from RIF. iTRAQ techniques based on isotope markers can identify and quantify low abundance proteomics, and may be suitable for identifying differentially expressed proteins in RIF. This study provides novel evidence that TPPP3 and HSD17B2 may be effective targets for the diagnosis and treatment of non-receptive endometrium and RIF.

Tppp3+ synovial/tendon sheath progenitor cells contribute to heterotopic bone after trauma

Heterotopic ossification (HO) is a pathological process resulting in aberrant bone formation and often involves synovial lined tissues. During this process, mesenchymal progenitor cells undergo endochondral ossification. Nonetheless, the specific cell phenotypes and mechanisms driving this process are not well understood, in part due to the high degree of heterogeneity of the progenitor cells involved. Here, using a combination of lineage tracing and single-cell RNA sequencing (scRNA-seq), we investigated the extent to which synovial/tendon sheath progenitor cells contribute to heterotopic bone formation. For this purpose, Tppp3 (tubulin polymerization-promoting protein family member 3)-inducible reporter mice were used in combination with either Scx (Scleraxis) or Pdgfra (platelet derived growth factor receptor alpha) reporter mice. Both tendon injury- and arthroplasty-induced mouse experimental HO models were utilized. ScRNA-seq of tendon-associated traumatic HO suggested that Tppp3 is an early progenitor cell marker for either tendon or osteochondral cells. Upon HO induction, Tppp3 reporter+ cells expanded in number and partially contributed to cartilage and bone formation in either tendon- or joint-associated HO. In double reporter animals, both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells gave rise to HO-associated cartilage. Finally, analysis of human samples showed a substantial population of TPPP3-expressing cells overlapping with osteogenic markers in areas of heterotopic bone. Overall, these data demonstrate that synovial/tendon sheath progenitor cells undergo aberrant osteochondral differentiation and contribute to HO after trauma.

#4996 TPPP3 REGULATES THE MICROTUBULE ACETYLATION AND PROMOTING YAP NUCLEAR RECRUITMENT IN HUMAN PODOCYTES

Abstract Background and Aims Minimal change disease (MCD) is a podocytopathy more commonly seen in children, but it also accounts for 10%–25% of adult NS. Patients with MCD manifest with abundant albuminuria, which is related to the damage of the glomerular filtration barrier. Podocytes are major components of the glomerular filtration barrier and play a crucial role in maintaining the integrity of the glomerular filtration barrier. Alterations in podocyte actin critically affect podocyte function. Tubulin polymerization-promoting protein family member 3 (TPPP3) is regulator of microtubule dynamic that has microtubule bundling activity. However, the mechanism of TPPP3 involved in podocyte apoptosis is remains unknown. Method Single-cell RNA sequencing comprehensively characterized the expression of TPPP3 in human glomeruli. We cultured human immortalized podocyte cells (HPC) in vitro, and constructed the HPC injury model. Immunofluorescence, western blot were used to determine the expression of TPPP3, α-tubulin, acetylated tubulin, phosopho-YAP1, Podocyte apoptosis was determined by Annexin V APC Apoptosis Detection Kit. Results TPPP3 was specifically expressed in human glomerular podocytes, and its expression level was significantly decreased in the cases of MCD and ADR induced podocyte injury(P&amp;lt;0.05). Knockdown of TPPP3 leaded to decreasing expression of acetylated tubulin in podocytes(P&amp;lt;0.05). Phosopho-YAP1 was also observed in the TPPP3 knockdown HPCs(P&amp;lt;0.05). Apoptosis was increased in the TPPP3 knockdown HPCs, suggesting TPPP3 may be related to the podocytes injury and apoptosis. Conclusion TPPP3 is involved with the apoptosis of podocytes by regulates nuclear recruitment of YAP which was promoted by microtubule acetylation.

P25alpha Domain-Containing Proteins of Apicomplexans and Related Taxa.

TPPP (tubulin polymerization promoting protein)-like proteins contain one or more p25alpha (Pfam05517) domains. TPPP-like proteins occur in different types as determined by their length (e.g., long-, short-, truncated-, and fungal-type TPPP) and include the protein apicortin, which possesses another domain, doublecortin (DCX, Pfam 03607). These various TPPP-like proteins are found in various phylogenomic groups. In particular, short-type TPPPs and apicortin are well-represented in the Myzozoa, which include apicomplexans and related taxa, chrompodellids, dinoflagellates, and perkinsids. The long-, truncated-, and fungal-type TPPPs are not found in the myzozoans. Apicortins are found in all apicomplexans except one piroplasmid species, present in several other myzozoans, and seem to be correlated with the conoid and apical complex. Short-type TPPPs are predominantly found in myzozoans that have flagella, suggesting a role in flagellum assembly or structure.

Somatic SNCA Copy Number Variants in Multiple System Atrophy are Related to Pathology and Inclusions.

Somatic α-synuclein (SNCA) copy number variants (CNVs, specifically gains) occur in multiple system atrophy (MSA) and Parkinson's disease brains. The aim was to compare somatic SNCA CNVs in MSA subtypes (striatonigral degeneration [SND] and olivopontocerebellar atrophy [OPCA]) and correlate with inclusions. We combined fluorescent in situ hybridization with immunofluorescence for α-synuclein and in some cases oligodendrocyte marker tubulin polymerization promoting protein (TPPP). We analyzed one to three brain regions from 24 MSA cases (13 SND, 11 OPCA). In a region preferentially affected in one subtype (putamen in SND, cerebellum in OPCA), mosaicism was higher in that subtype, and cells with CNVs were 4.2 times more likely to have inclusions. In the substantia nigra, nonpigmented cells with CNVs and TPPP were about six times more likely to have inclusions. The correlation between SNCA CNVs and pathology (at a regional level) and inclusions (at a single-cell level) suggests a role for somatic SNCA CNVs in MSA pathogenesis. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Early and extensive alterations of glial connexins, distal oligodendrogliopathy type demyelination, and nodal/paranodal pathology are characteristic of multiple system atrophy.

The pathological hallmark of multiple system atrophy (MSA) is aberrant accumulation of phosphorylated α-synuclein in oligodendrocytes, forming glial cytoplasmic inclusions (GCIs). Extensive demyelination occurs particularly in the olivopontocerebellar and striatonigral pathways, but its precise mechanism remains elusive. Glial connexins (Cxs), which form gap junction channels between astrocytes and oligodendrocytes, play critical roles in myelin maintenance, and have not been studied in MSA. Therefore, we immunohistochemically investigated glial Cx changes in the cerebellar afferent fibers in 15 autopsied patients with MSA. We classified demyelinating lesions into three stages based on Klüver-Barrera staining: early (Stage I), intermediate (Stage II), and late (Stage III) stages showing subtle, moderate, and severe myelin reduction, respectively. Myelin-associated glycoprotein, but not myelin oligodendrocyte glycoprotein, was preferentially decreased in Stage I, suggesting distal oligodendrogliopathy type demyelination. Accumulation of phosphorylated α-synuclein in oligodendrocytes was frequently seen in Stage I but less frequently observed in Stages II and III. Tubulin polymerization-promoting protein (TPPP/p25α)-positive oligodendrocytes were preserved in Stage I but successively decreased in Stages II and III. Even at Stage I, Cx32 was nearly absent from myelin, despite the relative preservation of other nodal proteins, such as neurofascin, claudin-11/oligodendrocyte-specific protein, and contactin-associated protein 1, which successively decreased in the later stages. Cx32 was re-distributed in the oligodendrocyte cytoplasm and co-localized with GCIs. Cx47 gradually decreased at the oligodendrocyte surface in a stage-dependent manner but was not co-localized with GCIs. Astrocytic Cx43 was down-regulated in Stage I but up-regulated in Stages II and III, reflecting astrogliosis. Cx43/Cx47 gap junctions significantly decreased from Stage I to III. Activated microglia/macrophages and T cells infiltrated in Stage I rather than Stages II and III. Therefore, early and extensive alterations of glial Cxs, particularly Cx32 loss, occur in MSA and may accelerate distal oligodendrogliopathy type demyelination and nodal/paranodal dysfunction through disruption of inter-glial communication.

OR24-1 Paracrine Growth Factor Signaling Regulates Muscle Regeneration in Aged Mice via microRNA 501

Abstract Background Skeletal muscle is one of to the most dynamic and plastic tissues. The capacity to regenerate arises from adult muscle stem cells termed myogenic progenitors (MPs) that are activated in response to injury and differentiate to replenish damaged muscle fibers. The age-related loss of muscle mass is reflected in the reduced regenerative potential of MPs. The underlying mechanisms are still unclear and insights into alterations in adult muscle stem cells during ageing are urgently needed. We have previously identified a novel muscle-specific microRNA, miR-501-3p, that is enriched in activated MPs. Pharmacological inhibition of miR-501 during muscle regeneration promoted small-diameter neofibers. Here, we provide evidence for the regulation of miR-501 in skeletal muscle during aging and its effect on newly formed myofibers using genetic deletion in mice. Methods We analysed the regulation and upstream signalling pathways of miR-501 in primary myoblasts from mice and humans and in skeletal muscle from young and aged mice. We generated a novel Cre-loxP mouse model, which allowed for global and MP-specific deletion of miR-501. Skeletal muscle regeneration was induced using cardiotoxin (CTX) and assessed using immunofluorescence and transcriptomic analysis. Results miR-501 was upregulated in muscle cells by the paracrine growth factors platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) through the Janus tyrosine kinase (JAK)-signal transducer and activator of transcription-3 (STAT3) signalling axis. Expression levels of miR-501 as well as its upstream regulators Pdgf and Vegf were significantly reduced in skeletal muscle from aged mice (22 months) compared to young mice (3 months) by 67.8% (p&amp;lt;0.001), 40.0% (p&amp;lt;0.01) and 36.3% (p&amp;lt;0.05), respectively. Genetic deletion of miR-501 in mice resulted in muscle fibers with decreased size in adult skeletal muscle and in newly formed myofibers during muscle regeneration. RNA-seq of regenerating muscle annotated a critical role for miR-501 in the organisation of muscle filaments. We identified a miR-501-dependent sarcomeric gene signature that involves genes such as myosin heavy chains, titin and tubulin polymerization-promoting protein. This was confirmed in vitro in myotubes from mice and humans in which miR-501 was deleted either genetically or pharmacologically using antagomirs. Altered sarcomeric gene expression correlated with an increased sarcomere length in myotubes lacking miR-501 (2.49 ± 0.08μm vs 3.22 ± 0.15μm, p&amp;lt;0.01). Conclusion Our data indicate that paracrine growth factor signalling contributes to muscle fiber formation via miR-501 in myogenic progenitor cells and its effect on the sarcomere. Restoring the Pdgf+Vegf/miR-501 axis might have the potential to improve muscle formation during aging. Presentation: Monday, June 13, 2022 11:00 a.m. - 11:15 a.m.

α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α

α-synucleinopathy is driven by an imbalance of synthesis and degradation of α-synuclein (αSyn), causing a build up of αSyn aggregates and post-translationally modified species, which not only interfere with normal cellular metabolism but also by their secretion propagates the disease. Therefore, a better understanding of αSyn degradation pathways is needed to address α-synucleinopathy. Here, we used the nerve growth factor-differentiated catecholaminergic PC12 neuronal cell line, which was conferred α-synucleinopathy by inducible expression of αSyn and tubulin polymerization-promoting protein p25α. p25α aggregates αSyn, and imposes a partial autophagosome-lysosome block to mimic aspects of lysosomal deficiency common in neurodegenerative disease. Under basal conditions, αSyn was degraded by multiple pathways but most prominently by macroautophagy and Nedd4/Ndfip1-mediated degradation. We found that expression of p25α induced strong p38MAPK activity. Remarkably, when opposed by inhibitor SB203580 or p38MAPK shRNA knockdown, endolysosomal localization and degradation of αSyn increased, and αSyn secretion and cytotoxicity decreased. This effect was specifically dependent on Hsc70 and the endosomal sorting complex required for transport machinery, but different from classical microautophagy, as the αSyn Hsc70 binding motif was unnecessary. Furthermore, in a primary neuronal (h)-αSyn seeding model, p38MAPK inhibition decreased pathological accumulation of phosphorylated serine-129-αSyn and cytotoxicity. In conclusion, p38MAPK inhibition shifts αSyn degradation from various forms of autophagy to an endosomal sorting complex required for transport-dependent uptake mechanism, resulting in increased αSyn turnover and cell viability in p25α-expressing cells. More generally, our results suggest that under conditions of autophagolysosomal malfunction, the uninterrupted endosomal pathway offers a possibility to achieve disease-associated protein degradation.

The expression and role of tubulin polymerization-promoting protein 3 in oral squamous cell carcinoma

ObjectiveTo study the expression, prognostic landscape, and the function of tubulin polymerization-promoting protein 3 (TPPP3) in oral squamous cell carcinoma (OSCC) and explore the relationship between TPPP3 expression and immune infiltration. DesignImmunohistochemical analysis using tissue microarray of 65 OSCC samples and 10 normal oral mucosa samples was performed to explore the expression levels in OSCC. 5 OSCC samples and 5 adjacent normal oral mucosa samples were selected to further analyze the expression levels by western blot. Some online bioinformatics analysis tools were adopted to study the transcriptome and prognostic landscape of TPPP3, the relationship of TPPP3 expression with immune infiltration, and the most possible “functions and pathways” of TPPP3 in head and neck squamous cell carcinoma (HNSC). ResultsImmunohistochemical, western blot and bioinformatics analysis showed that the expression of TPPP3 in OSCC was significantly lower than that in the normal oral mucosa. High expression of TPPP3 was associated with a good prognosis of HNSC. TPPP3 expression was significantly positively correlatedwith infiltration level of B cell plasma and myeloid dendritic cells activated in HNSC, while it was negatively correlated with infiltration level of Tregs cells, CD8+ T cells, B cell memory. Pathway analysis results revealed one most probable function and two significant pathways in which TPPP3 participates. ConclusionsTPPP3 can serve as a potential diagnostic biomarker and a prognostic protective indicator of OSCC. It plays an important role in the recruitment and regulation of tumor-infiltrating immune cells, which may affect the survival of patients.

Nogo-A Induced Polymerization of Microtubule Is Involved in the Inflammatory Heat Hyperalgesia in Rat Dorsal Root Ganglion Neurons.

The microtubule, a major constituent of cytoskeletons, was shown to bind and interact with transient receptor potential vanilloid subfamily member 1 (TRPV1), and serves a pivotal role to produce thermal hyperalgesia in inflammatory pain. Nogo-A is a modulator of microtubule assembly and plays a key role in maintaining the function of TRPV1 in inflammatory heat pain. However, whether the microtubule dynamics modulated by Nogo-A in dorsal root ganglion (DRG) neurons participate in the inflammatory pain is not elucidated. Here we reported that the polymerization of microtubules in the DRG neurons, as indicated by the acetylated α-tubulin, tubulin polymerization-promoting protein 3 (TPPP3), and microtubule numbers, was significantly elevated in the complete Freund’s adjuvant (CFA) induced inflammatory pain. Consistent with our previous results, knock-out (KO) of Nogo-A protein significantly attenuated the heat hyperalgesia 72 h after CFA injection and decreased the microtubule polymerization via up-regulation of phosphorylation of collapsin response mediator protein 2 (CRMP2) in DRG. The colocalization of acetylated α-tubulin and TRPV1 in DRG neurons was also reduced dramatically in Nogo-A KO rats under inflammatory pain. Moreover, the down-regulation of TRPV1 in DRG of Nogo-A KO rats after injection of CFA was reversed by intrathecal injection of paclitaxel, a microtubule stabilizer. Furthermore, intrathecal injection of nocodazole (a microtubule disruptor) attenuated significantly the CFA-induced inflammatory heat hyperalgesia and the mechanical pain in a rat model of spared nerve injury (SNI). In these SNI cases, the Nogo-A and acetylated α-tubulin in DRG were also significantly up-regulated. We conclude that the polymerization of microtubules promoted by Nogo-A in DRG contributes to the development of inflammatory heat hyperalgesia mediated by TRPV1.

Gain of TPPP as a predictor of progression in patients with bladder cancer.

The present study investigated the role of tubulin polymerization promoting protein (TPPP) in the regulation of bladder cancer (BC) cell proliferation and migration, in addition to the association between TPPP gene copy number amplification and clinicopathological characteristics of BC. TPPP gene amplification was measured in human BC epithelial cells and samples obtained from 52 patients with BC via fluorescence in situ hybridization. TPPP gain was defined as mean TPPP copy number >2.2 per nucleus (cutoff). The neutrophil-to-lymphocyte ratio (NLR) was also obtained from the preoperative data of the patients. For in vitro assays, BC cell lines were transfected with either TPPP small interfering RNAs or scrambled control, following which cell proliferation and migration were determined using Cell Counting Kit-8 and Transwell migration assays, respectively. The percentage of cells with TPPP copy number amplification in the four BC epithelial cell lines (MGH-U1, -U1R, -U3, -U4) examined (86.0-100.0%) was found to be higher compared with that in the normal human uroepithelial cell lines (3.0 and 9.0%). Patients were divided into one- (1.9%), two- (55.8%), three- (7.7%), four- (26.9%) and five-copy (7.7%) types. Results calculated using Fisher's exact test indicated that the gain of TPPP in patients with BC associated significantly with age (P<0.05), advanced histological grade (P<0.001), tumor stage (P<0.05), histological type (P<0.001) and NLR (P<0.05). In MGH-U1R and MGH-U4 cells, cell proliferation and migration were revealed to be significantly lower following TPPP knockdown compared with those in cells transfected with the scrambled control. In conclusion, findings from the present study suggest that TPPP is important for cell proliferation, cell migration and BC progression, such that TPPP copy number assessment would be advised for preoperative urine cytology for urothelial neoplasia diagnosis.

Drosophila tubulin polymerization promoting protein mutants reveal pathological correlates relevant to human Parkinson\u2019s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disorder with no known cure. PD is characterized by locomotion deficits, nigrostriatal dopaminergic neuronal loss, mitochondrial dysfunctions and formation of α-Synuclein aggregates. A well-conserved and less understood family of Tubulin Polymerization Promoting Proteins (TPPP) is also implicated in PD and related disorders, where TPPP exists in pathological aggregates in neurons in patient brains. However, there are no in vivo studies on mammalian TPPP to understand the genetics and neuropathology linking TPPP aggregation or neurotoxicity to PD. Recently, we discovered the only Drosophila homolog of human TPPP named Ringmaker (Ringer). Here, we report that adult ringer mutants display progressive locomotor disabilities, reduced lifespan and neurodegeneration. Importantly, our findings reveal that Ringer is associated with mitochondria and ringer mutants have mitochondrial structural damage and dysfunctions. Adult ringer mutants also display progressive loss of dopaminergic neurons. Together, these phenotypes of ringer mutants recapitulate some of the salient features of human PD patients, thus allowing us to utilize ringer mutants as a fly model relevant to PD, and further explore its genetic and molecular underpinnings to gain insights into the role of human TPPP in PD.

Regulation of the tubulin polymerization-promoting protein by Ca2+/S100 proteins

To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111–160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization.

TPPP3 inhibits the proliferation, invasion and migration of endometrial carcinoma targeted with miR-1827.

Database screening indicated that tubulin polymerization-promoting protein 3 (TPPP3) was involved in pathogenesis of multiple cancer types. miR-1827 has a potential role in a variety of human cancers. However, the role of TPPP3 and its underlying molecular mechanism in endometrial cancer (EC) has not been investigated. Herein, we aimed to reveal the role of TPPP3/miR-1827 in EC progression. Tumour tissue and whole blood samples were collected for the detection of TPPP3 expression. TPPP3 shRNAs and pcDNA-TPPP3 were applied to knockdown or upregulate the TPPP3 expression, and miR-1827 mimic was used to upregulate miR-1827 level. CCK-8 and colony assays were applied to estimate the cell proliferation. Wound healing and Transwell assays were conducted to assess the cell migration and invasion abilities. The dual-luciferase reporter assay was conducted to validate the putative binding site between TPPP3 and miR-1827. Expression of TPPP3, miR-1827 and related proteins in cell lines, tissue and whole blood sample were detected using western blot, RT-qPCR and immunofluorescence. TPPP3 was observed markedly elevated in EC patients and cells. TPPP3 knockdown displayed evident suppression in cell proliferation, migration and invasion in vitro and in vivo. Moreover, we identified TPPP3 as a direct and functional target gene of miR-1827 in EC cells. The miR-1827 induced regulatory effects on EC cells were partially reversed by TPPP3. Additionally, in vivo study confirmed the findings discovered in vitro. TPPP3 exerted oncogenic roles in EC progression by sponging miR-1827. This finding might provide potential targets for EC therapy.

SIRT1 decelerates morphological processing of oligodendrocyte cell lines and regulates the expression of cytoskeleton-related oligodendrocyte proteins

SIRT1 is involved in the regulation of a variety of biological processes such as metabolism, stress response, autophagy and differentiation. Although progenitor cells of oligodendrocytes (OPCs) express high level of SIRT1, its function on differentiation is unknown. Because we have shown that SIRT1 plays a pivotal role in differentiation of neural precursor cells, we hypothesized that SIRT1 may also participate in the differentiation of oligodendrocytes (OLGs). We examined whether SIRT1 was expressed in two human oligodendrocyte cell lines: KG-1-C and MO 3.13 OLG. Transfection of cell lines with SIRT1-siRNA and SIRT2-siRNA promoted the extension of cellular processes. SIRT1-siRNA and SIRT2-siRNA increased acetyl-α-tubulin level, conversely, over expression of SIRTs resulted in decreased the ratio of acetyl-α-tubulin to α-tubulin. We also found knockdown of SIRT1 and SIRT2 induced overexpression of βIV-tubulin and tubulin polymerization promoting protein (TPPP) (OLG-specific cytoskeleton-related molecules) that distributed widely in cell bodies. Taken together, SIRT1 may play a role in oligodenroglial differentiation and myelinogenesis.

Characterization of the structure, vascularity, and stem/progenitor cell populations in porcine Achilles tendon (PAT).

This study aimed to characterize porcine Achilles tendon (PAT) in terms of its structural components, vascularity, and resident tendon cells. We found that PAT is composed of a paratenon sheath, a core of fascicles, and an endotenon/interfascicular matrix (IFM) that encases the fascicle bundles. We analyzed each of these three tendon components structurally using tissue sections and by isolating cells from each component and analyzing in vitro. Many blood vessel-like tissues were present in the paratenon and IFM but not in fascicles, and the vessels in the paratenon and IFM appeared to be inter-connected. Cells isolated from the paratenon and IFM displayed characteristics of vascular stem/progenitor cells expressing the markers CD105, CD31, with α-smooth muscle actin (α-SMA) localized surrounding blood vessels. The isolated cells from paratenon and IFM also harbored abundant stem/progenitor cells as evidenced by their ability to form colonies and express stem cell markers including CD73 and CD146. Furthermore, we demonstrate that both paratenon and IFM-isolated cells were capable of undergoing multi-differentiation. In addition, both paratenon and IFM cells expressed elastin, osteocalcin, tubulin polymerization promoting protein (TPPP), and collagen IV, whereas fascicle cells expressed none of these markers, except collagen I. The neurotransmitter substance P (SP) was also found in the paratenon and IFM-localized surrounding blood vessels. The findings of this study will help us to better understand the vascular and cellular mechanisms of tendon homeostasis, injury, healing, and regeneration.