TUBB3 Research Articles

Intravitreal S100B Injection Leads to Progressive Glaucoma Like Damage in Retina and Optic Nerve

The glial protein S100B, which belongs to a calcium binding protein family, is up-regulated in neurological diseases, like multiple sclerosis or glaucoma. In previous studies, S100B immunization led to retinal ganglion cell (RGC) loss in an experimental autoimmune glaucoma (EAG) model. Now, the direct degenerative impact of S100B on the retina and optic nerve was evaluated. Therefore, 2 μl of S100B was intravitreally injected in two concentrations (0.2 and 0.5 μg/μl). At day 3, 14 and 21, retinal neurons, such as RGCs, amacrine and bipolar cells, as well as apoptotic mechanisms were analyzed. Furthermore, neurofilaments, myelin fibers and axons of optic nerves were evaluated. In addition, retinal function and immunoglobulin G (IgG) level in the serum were measured. At day 3, RGCs were unaffected in the S100B groups, when compared to the PBS group. Later, at days 14 and 21, the RGC number as well as the β-III tubulin protein level was reduced in the S100B groups. Only at day 14, active apoptotic mechanisms were noted. The number of amacrine cells was first affected at day 21, while the bipolar cell amount remained comparable to the PBS group. Also, the optic nerve neurofilament structure was damaged from day 3 on. At day 14, numerous swollen axons were observed. The intraocular injection of S100B is a new model for a glaucoma like degeneration. Although the application site was the eye, the optic nerve degenerated first, already at day 3. From day 14 on, retinal damage and loss of function was noted. The RGCs in the middle part of the retina were first affected. At day 21, the damage expanded and RGCs had degenerated in all areas of the retina as well as amacrine cells. Furthermore, elevated IgG levels in the serum were measured at day 21, which could be a sign of a late and S100B independet immune response. In summary, S100B had a direct destroying impact on the axons of the optic nerve. The damage of the retinal cell bodies seems to be a consequence of this axon loss.

Morphological and quantitative analysis on α-tubulin modifications in glioblastoma cells

Glioblastoma is one of the primary tumors of the brain with high invasiveness and lethality. In the study of its pathophysiology in recent years much attention has been paid to the microtubular network, but exclusively to β-III tubulin, whose presence in glioblastoma cells is associated with the degree of malignancy and diffusion. As is well known, the microtubular network performs its many functions thanks to various post-translational modifications, most of which affect the α-tubulin subunit. These modifications, able to coexist in the same microtubule, bind certain driving and cargo proteins, deeply influencing cellular functions. Since there are no data in the literature about the diverse post-translational modifications of tubulin in glioblastoma cells, this work aims to fill this gap. In the present work, through immunofluorescence, morphological analysis and Western blot, we studied the pattern of tyrosinated, detyrosinated, delta 2 (Δ2), acetylated and polyglutamylated tubulin. We detected good immunopositivity in fluorescence for almost all the modifications examined. Only Δ2 showed a very low signal. Western blot displayed that the most abundant tubulin modifications in glioblastoma cells were tyrosinated, acetylated and polyglutamylated. Morphological evaluation revealed that these modifications were more present along the cytoplasmic extensions, less evident around the cell body and always strongly evident in the mitotic spindles of the dividing cells. For the first time, the most abundant post-translational modifications and their cellular compartmentalization in glioblastoma cells have been highlighted, suggesting a novel approach in the study of their microtubular network and in the search of new experimental therapeutic strategies.

Sterically induced conformational restriction: Discovery and preclinical evaluation of novel pyrrolo[3,2-d]pyrimidines as microtubule targeting agents.

The discovery, synthesis and biological evaluations of a series of nine N5-substituted-pyrrolo[3,2-d]pyrimidin-4-amines are reported. Novel compounds with microtubule depolymerizing activity were identified. Some of these compounds also circumvent clinically relevant drug resistance mechanisms (expression of P-glycoprotein and βIII tubulin). Compounds 4, 5, and 8-13 were one to two-digit nanomolar (IC50) inhibitors of cancer cells in culture. Contrary to recent reports (Banerjee et al. J. Med. Chem.2018, 61, 1704-1718), the conformation of the most active compounds determined by 1H NMR and molecular modeling are similar to that reported previously and in keeping with recently reported X-ray crystal structures. Compound 11, freely water soluble as the HCl salt, afforded statistically significant inhibition of tumor growth in three xenograft models [MDA-MB-435, MDA-MB-231 and NCI/ADR-RES] compared with controls. Compound 11 did not display indications of animal toxicity and is currently slated for further preclinical development.

AB084. intracavernous administration of embryonic stem cell-derived exosome restores erectile function through penile neurovascular regeneration in the streptozotocin-induced diabetic mouse

BackgroundExosome contains a variety of protein, mRNA, and miRNA and is known to play an important role in intercellular communication as a bio-nanoparticle with a diameter of 40 to 100 nm. Recent studies have demonstrated the therapeutic potential of exosome in a variety of animal models for cardiovascular diseases and neuropathies. The aim of this study was to investigate effectiveness of embryonic stem cell (ESC)-derived exosome in restoring erectile function in diabetic mice.MethodsDiabetes was induced by intraperitoneal injection of streptozotocin into 8-week-old C57BL/6 male mice. At 8 weeks after the induction of diabetes, the animals were distributed into 3 groups: control nondiabetic mice and diabetic mice receiving two successive intracavernous injections of phosphate buffered saline (PBS) (days −3 and 0; 20 µL) or ESC-derived exosome (days −3 and 0; 1 µg in 20 µL of PBS). Two weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested and stained with antibodies to PECAM-1, smooth muscle α-actin, NG2, and βIII tubulin. We also determined angiogenic potential of ESC-derived exosome in an ex vivo aortic ring assay and in primary cultured mouse cavernous endothelial cell (MCEC) and pericyte (MCP) mono-culture or co-culture system in vitro.ResultsIntracavernous injections of ESC-derived exosome significantly improved erectile function in diabetic mice, which reached up to 90% of control values. ESC-derived exosome induced significant restoration of cavernous contents of endothelial cells, smooth muscle cells, pericytes, and neuronal cells in diabetic condition. Moreover, ESC-derived exosome promoted microvascular sprouting from aortic ring and accelerated tube formation in primary cultured MCEC and MCP mono-culture or co-culture system in vitro.ConclusionsESC-derived exosome successfully restored erectile function through enhanced cavernous angiogenesis and neural regeneration in diabetic mice. Further studies are needed to clarify mechanism by which ESC-derived exosome induces neurovascular repair.

Age-related loss of VGLUT1 excitatory, but not VGAT inhibitory, immunoreactive terminals on motor neurons in spinal cords of old sarcopenic male mice

Age-related changes in ventral lumbar spinal cord (L3-L5) were compared in young [3month, (M)] and old (27M) C57BL/6J male mice. The aged mice had previously been shown to exhibit sarcopenia and changes to peripheral nerve morphology. The putative connectivity of β-III tubulin positive α-motor neurons was compared in immunostained transverse sections using excitatory and inhibitory terminal markers vesicular glutamate transporter-1 (VGLUT1) and vesicular GABA transporter (VGAT). Glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) immunostaining was used to monitor changes in astrocyte and microglial phenotype respectively. For a given motor neuron, the neuronal perimeter was outlined and terminals immunoreactive for VGLUT1 or VGAT in close apposition to the soma were identified. By 27M, the percentage coverage and total number of VGLUT1 immunoreactive terminals immediately adjacent to the soma of α-motor neurons was significantly decreased compared with young mice. However, percentage coverage of immunoreactive VGAT inhibitory terminals did not change significantly with age. The gray matter of 27M spinal cords showed increased astrocytic and microglial activity. The loss of VGLUT1 terminals on α-motor neurons, terminals known to be derived from proprioceptive muscle afferents, may further impair sensorimotor control of hind limb skeletal muscle function in old mice.

Stemness and Regenerative Potential of Corneal Stromal Stem Cells and Their Secretome After Long-Term Storage: Implications for Ocular Regeneration.

PurposeTo assess the stemness and regenerative potential of cryopreserved corneal stromal stem cells (cryo-CSSCs) after long-term storage. We also used the secretome from these cells to observe the effect on wound-healing capacity of corneal fibroblasts and on the expression of fibrotic markers during wound healing.MethodsCSSCs were obtained from three donors and stored in liquid nitrogen for approximately 10 years. Post thaw, cryo-CSSCs were characterized for stemness using phenotypic and genotypic markers along with colony-forming efficiency and three-dimensional spheroid formation. Multilineage differentiation was observed by differentiation into osteocytes, adipocytes, neural cells, and keratocytes. Secretome was harvested by culturing cryo-CSSCs in log phase. Wound-healing capacity was observed by live-cell time-lapse microscopy. Statistical analysis was done using 1-way ANOVA and Tukey posttest.ResultsCSSCs displayed good viability post thaw and showed >90% expression of stem cell markers CD90, CD73, CD105, STRO1, and CD166. cryo-CSSCs also expressed stem cell genes OCT4, KLF4, and ABCG2, and could also form colonies and three-dimensional spheroids. Multipotency assessment showed that all three cryo-CSSCs could differentiate into osteocytes, adipocytes, neural cells, as shown by β-III tubulin and neurofilament antibody staining and corneal keratocytes as observed by staining for Kera C, J19, and collagen V antibodies. The secretome derived from these three populations could promote the wound healing of corneal fibroblasts and reduce the expression of fibrotic markers SPARC and fibronectin.ConclusionsCSSCs maintained their stemness and multipotency after long-term storage, and secretome derived from these cells can be of paramount importance for corneal regeneration and prevention of fibrosis.

A Novel Cell Penetrating Peptide for the Differentiation of Human Neural Stem Cells.

Retinoic acid (RA) is a bioactive lipid that has been shown to promote neural stem cell differentiation. However, the highly hydrophobic molecule needs to first solubilize and translocate across the cell membrane in order to exert a biological response. The cell entry of RA can be aided by cell penetrating peptides (CPPs), which are short amino acid sequences that are able to carry bioactive cargo past the cell membrane. In this work, a novel cell penetrating peptide was developed to deliver RA to human neural stem cells and, subsequently, promote neuronal differentiation. The novel CPP consists of a repeating sequence, whose number of repeats is proportional to the efficiency of cell penetration. Using fluorescence microscopy, the mode of translocation was determined to be related to an endocytic pathway. The levels of β-III tubulin (Tubb3) and microtubule associated protein 2 (MAP2) expression in neural stem cells treated with RA conjugated to the CPP were assessed by quantitative immunocytochemistry.

Abstract 2663: CR42-24, a novel colchicine derivative, a therapeutic for bladder cancer

Abstract Colchicine is an anti-mitotic drug that targets unpolymerized tubulin, and inhibits microtubule polymerization. It is primarily used to treat gout but has been investigated in numerous clinical trials to treat conditions such as leukemia (ALL), prostate cancer, Behcet's disease, etc.. However, due to its narrow therapeutic window colchicine has had limited clinical translation. A possible approach to reduce general is to produce a derivative with an increased cancer specificity and selectivity by a narrower molecular target selection. Previously, our lab has designed and synthesized a novel colchicine derivative (CR42-24) with a more favorable pharmacological profile compared to colchicine. This was accomplished by designing a structure with an increased affinity for βIII tubulin. βIII tubulin is a β-tubulin isotype that is incorporated into tubulin dimers that make up microtubules. βIII is an excellent target for novel therapies as it has low expression in healthy tissue, is overexpressed in metastatic cancers, and is a clinical marker of poor prognosis. Using cell line screening we demonstrate that CR42-24 is highly toxic to a variety of cancer types with IC50 values at low nanomolar concentrations. More specifically CR42-24 is shown to be highly effective against bladder cancer. Current chemotherapy for bladder cancer is a combination of gemcitabine and cisplatin (Gem/Cis). Although marginally successful, many patients develop resistance to Gem/Cis leaving them with limited options for a second line therapy, thus development of alternative therapies is highly desired. Using in vitro assays we demonstrate that CR42-24 kills aggressive bladder cancer cell types, and is more effective than gemcitabine and cisplatin. Additionally, CR42-24 doubles survival rates of mice with bladder cancer xenografts. We also show that CR42-24 is highly synergistic with other chemotherapies, thereby increasing its therapeutic potential. Through our studies we have shown that CR42-24 is effective in treating aggressive bladder cancer and thus may serve as an alternative or second line therapy. Citation Format: Clayton Bell, Kyle Potts, Desmond Pink, John Lewis, Jack Tuszynski. CR42-24, a novel colchicine derivative, a therapeutic for bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2663.

Abstract 4851: Potent cell cycle inhibitors suitable for treatment of multidrug-resistant tumors

Abstract We have developed a class of small-molecule cytotoxic compounds that are highly potent in an in vitro tubulin polymerization assay. Preclinical studies show that these drug candidates are potent against a range of cancer cell lines and may be well suited for the treatment of hematologic or solid tumors resistant to existing chemotherapy agents. We will present data on representative members of this class that inhibit cancer cell growth at nanomolar concentrations and are potent against a variety of cancer cell lines, including liver, breast, ovarian, and lung. The compounds induce cell cycle arrest in the G2M state within 24 hours with cell death occurring over multiple days. These tubulin inhibitors also behave as potent antiangiogenesis agents and inhibit endothelial tube formation in HUVEC cells at nanomolar concentrations. Functional assays using efflux pump inhibitors show that, compared to many major chemotherapy agents, our class of drug candidates is significantly less affected by the efflux pumps MDR1 and MRP1, which are commonly overexpressed as a mechanism of multidrug resistance. Our compounds show comparable potency against wild-type and cell lines developed to be resistant to other cancer drugs, while the potency of clinically relevant compounds such as paclitaxel or doxorubicin is reduced between 80x and about 3000x in the same assay. This feature suggests that our tubulin inhibitors may be active against tumors that are resistant to common cancer drugs. Overexpression of the β-III isotype of tubulin, which is known to occur in many aggressive and metastatic tumors, is another clinically relevant mechanism of resistance to microtubule-targeting anticancer agents. It has been correlated with significantly lower response to docetaxel-based chemotherapy in a number of cancers and is considered an indicator of resistance to paclitaxel and vinorelbine. We will present preclinical data showing that our compounds have similar activity against cells showing normal levels and those highly overexpressing β-III tubulin. The compounds display good in vitro physicochemical properties as well as favorable in vivo pharmacokinetics. In addition, we will present in vivo tolerability and efficacy data in mice. The ability of this class of tubulin inhibitors to maintain their efficacy across multiple drug-resistant cancer cell lines makes them attractive candidates for development as chemotherapy agents. In particular, a new anticancer agent that is less susceptible to major transporters and retains its potency when β-III tubulin is overexpressed could lead to more effective precision second-line therapy. Citation Format: Mohan Sivaraja, Sivan Sizikov, Nilantha Sirisoma, Tamari Kirtadze, Madhuri Chattopadhyay, Makena Ewald, Subhadra Dash, Anne Wong, Georg Neckermann, Elaine To, Stephanie Chang, Timothy P. Shiau, David C. Williams, Kevin M. Short, Angels Estiarte, Anirban Datta, David B. Kita. Potent cell cycle inhibitors suitable for treatment of multidrug-resistant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4851.

Abstract 3549: Exosome-mediated transfer of sh-CD99 is sufficient to modulate cell differentiation in Ewing sarcoma

Abstract Background Ewing sarcoma (EWS) is an aggressive childhood bone tumor with still highly unmet clinical needs. The tumor is characterized by the fusion oncoprotein EWS-FLI1 and the high expression of the membrane glycoprotein CD99. CD99 has been found to be released by EWS cells linked to exosomes (EXOs), small vesicles able to influence cellular behavior and surrounding microenvironment by transferring their contents into nearby or distant recipient cells. The delivery of EXOs devoid of CD99 was sufficient to induce neural differentiation in recipient EWS cells through miR- 34a-dependent inhibition of Notch-NF-kB signaling, thus indicating a possible therapeutic potential. Aims The study has the ambition to: 1. Evaluate the capabilities of EXOs released by Ewing's sarcoma, to influence cancer growth and/or metastasis through the horizontal transfer of their cargos. 2. Characterize EXOs secreted by CD99-silenced EWS cells in terms of miRNAs and protein and evaluate their general antimetastatic function, if any, taking advantage of their capabilities to be transferred into a broad panel of different Ewing's sarcoma cell lines, as recipients. Results In the present study we identified the role of Ewing sarcoma-derived EXOs as mediators of signals involved in cancer growth, metastases and differentiation. In particular, we analyzed the ability of EXOs, expressing or not CD99, to modulate the phenotype of EWS cells. Delivery of EXOs from CD99-silenced cells was sufficient to induce neural differentiation in recipient EWS cells by neurite outgrowth and increased expression of β-III tubulin marker of neural differentiation accompanied in vitro by a significant reduction of proliferation, invasion and chemotaxis convey the same phenotype obtained by stable CD99 silencing. Moreover, microarray analysis revealed a signature of 56 miRNAs differentially expressed in EXOs derived from CD99-silenced cells compared to EWS-derived EXOs. We focused our attention on miR-214 and miR-199. Those miRNAs are involved in the regulation of CD99 itself like, EWS-FLI1 or CD99-mediated pathways related to cytoskeleton and membrane organization. As a next step we searched for any tumorigenic effect possibly associated with miR-199 and mir-214. The restored expression of miR-199 and miR-214 in two Ewing Sarcoma cell lines was accompanied by a significant reduction of proliferation, invasion and chemotaxis in vitro. The outcomes deriving from miR-199 and miR-214 enforced expression were also evaluated on capability of forming foci in agar semisolid medium. Results showed a significant decrease of the number of foci in both cell lines. Conclusions Exosomes deprived of CD99 can alter the balance of important cellular components necessary for the metastatic process of EWS cells, thus representing a novel exciting therapeutic possibility for the design of novel therapy against metastatic EWS. AIRC IG18451 to KS; IG18815 to AC. Citation Format: Alessandra De Feo, Marika Sciandra, Federica Felicetti, Manuela Ferracin, Alessandra Carè, Katia Scotlandi. Exosome-mediated transfer of sh-CD99 is sufficient to modulate cell differentiation in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3549.

5-FU resistant colorectal cancer cells possess improved invasiveness and βIII-tubulin expression

Purpose: Elevated b III -tubulin levels are associated with resistance to a broad spectrum of drugs in different carcinomas and are associated with poor prognosis of different epithelial cancers. 5-Fluorouracil (5-FU) is a widely used standard drug in chemotherapeutic regimens for colorectal cancer treatment, although the resistance to 5-FU is a major obstacle to successful therapy. The aim of the study was to compare the invasive and adhesion properties and the expression levels of b III -tubulin in a 5-Fluorouracil (5-FU)-resistant colorectal cancer (CRC) cell line HCT116 and parental cells. Methods: The 5-FU-resistant cell line was established by continuous stepwise selection with increasing concentrations of 5-FU. Cell viability and properties were evaluated using MTT, adhesion and transwell invasion assays respectively. The expression of b III -tubulin was revealed by immunoblot and immunofluorescence. Results: The derivative line is 25-fold resistant to 5-FU and characterized by altered cell morphology, twice as many cells of the 5-FU-resistant line fail to adhere than is the case for the parental cell line and were characterized by enhanced invasiveness, accompanied by increased b III -tubulin expression. In addition, we found that loss of b III -tubulin expression was correlated with loss of 5-FU resistance. Conclusion: Our results indicate that even though 5-FU does not target microtubules, there appears to be a correlation between b III -tubulin expression and resistance to 5-FU and that this is particularly important with regard to invasiveness. These findings indicate a possible contribution of b III -tubulin to 5-FU resistance in vivo.

The Efficacy of Graphene Foams for Culturing Mesenchymal Stem Cells and Their Differentiation into Dopaminergic Neurons.

The implantation of stem cells in vivo is the ideal approach for the restoration of normal life functions, such as replenishing the decreasing levels of affected dopaminergic (DA) neurons during neurodegenerative disease conditions. However, combining stem cells with biomaterial scaffolds provides a promising strategy for engineering tissues or cellular delivery for directed stem cell differentiation as a means of replacing diseased/damaged tissues. In this study, mouse mesenchymal stem cells (MSCs) were differentiated into DA neurons using sonic hedgehog, fibroblast growth factor, basic fibroblast growth factor, and brain-derived neurotrophic factor, while they were cultured within collagen-coated 3D graphene foams (GF). The differentiation into DA neurons within the collagen-coated GF and controls (collagen gels, plastic) was confirmed using β-III tubulin, tyrosine hydroxylase (TH), and NeuN positive immunostaining. Enhanced expression of β-III tubulin, TH, and NeuN and an increase in the average neurite extension length were observed when cells were differentiated within collagen-coated GF in comparison with collagen gels. Furthermore, these graphene-based scaffolds were not cytotoxic as MSC seemed to retain viability and proliferated substantially during in vitro culture. In summary, these results suggest the utility of 3D graphene foams towards the differentiation of DA neurons from MSC, which is an important step for neural tissue engineering applications.

Intrafamiliar clinical variability of circumferential skin creases Kunze type caused by a novel heterozygous mutation of N‐terminal TUBB gene

Circumferential skin creases Kunze type (CSC-KT; OMIM 156610, 616734) is a rare disorder characterized by folding of excess skin, which leads to ringed creases, known as Michelin Tire Baby Syndrome (MTBS). CSC-KT patients also exhibit facial dysmorphism, growth retardation, intellectual disability (ID) and multiple congenital malformations. Recently, 2 heterozygous mutations in TUBB gene and 4 mutations (both homozygous and heterozygous) in MAPRE2 gene were identified in 3 and 4 CSC-KT patients, respectively. In the 3 TUBB gene-related CSC-KT patients, all mutations fall in the N-terminal gene domain and were de novo. Mutations in the C-terminal of TUBB gene have been associated to microcephaly and structural brain malformation, in the absence of CSC-KT features. We report a 9-year-old boy with a diagnosis of CSC-KT based on MTBS, facial dysmorphism, microcephaly, severe ID, cortical atrophy and corpus callosum hypoplasia. Sanger sequencing identified a novel heterozygous c.218T>C (p.Met73Thr) mutation in the N-terminal of TUBB gene, that was inherited from the mother affected by isolated MTBS. This is the first report of inherited TUBB gene-related CSC-KT resulting from a novel heterozygous mutation in the N-terminal domain. Present data support the role of TUBB mutations in CSC-KT and definitely includes CSC-KT syndrome within the tubulinopathies.

Role of N‐type Calcium Channels in inflammatory pain and associated sensory nerve growth

Voltage‐gated calcium channels (VGCC) play an important role in pain neurotransmission by releasing neuropeptides substance P and calcitonin gene‐related peptide from the sensory neurons. Here, we identified a new role of VGCC in pain signaling by sensory nerve growth at the periphery in the skin. In the present study, we first assessed the role of N‐type calcium channels (Cav2.2) in inflammatory pain. Complete Freund's Adjuvant (CFA), a known inflammogen was injected into the hind‐paws of C57Bl6 mice to induce inflammation and dorsal root ganglia from the lumbar region (L4–L6) were isolated and cultured 24‐hour post injection. Voltage clamp recordings revealed a significant increase in Cav2.2 currents between contralateral and ipsilateral DRGs. Current clamp analysis of these DRGs showed a significant increase in action potential frequency upon CFA injection. A significant increase in Cav2.2 currents was also observed when CFA (1:1000) was directly added to DRG culture. The increase in Cav2.2 currents was significantly decreased upon addition of ω‐conotoxin (1 μM) into DRG culture. Immunocytochemical analysis of cultured DRGs with CFA (1:1000) revealed a significant increase in TRPV1, Cav2.2 and neuronal filament markers such as Synaptophysin and neuronal marker β‐III tubulin (TUJ1), indicate increased neuronal synthesis and neurite formation at the periphery. This was further corroborated by Western Blot analysis. Altogether, our study suggests a possible role of VGCC‐dependent sensory nerve growth that might be responsible for increased sensitivity to minimal stimuli in chronic pain.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Secretome of Differentiated PC12 Cells Enhances Neuronal Differentiation in Human Mesenchymal Stem Cells Via NGF-Like Mechanism

The secretome-mediated responses over cellular physiology are well documented. Stem cells have been ruling the field of secretomics and its role in regenerative medicine since the past few years. However, the mechanistic aspects of secretome-mediated responses and the role of other cells in this area remain somewhat elusive. Here, we investigate the effects of secretome-enriched conditioned medium (CM) of neuronally differentiated PC12 cells on the neuronal differentiation of human mesenchymal stem cells (hMSCs). The exposure to CM at a ratio of 1:1 (CM: conditioned medium of PC12 cells) led to neuronal induction in hMSCs. This neuronal induction was compared with a parallel group of cells exposed to nerve growth factor (NGF). There was a marked increase in neurite length and expression of neuronal markers (β-III tubulin, neurofilament-M (NF-M), synaptophysin, NeuN in exposed hMSCs). Experimental group co-exposed to NGF and CM showed an additive response via MAPK signaling and directed the cells particularly towards cholinergic lineage. The ability of CM to enhance the neuronal properties of stem cells could aid in their rapid differentiation into neuronal subtypes in case of stem cell transplantation for neuronal injuries, thus broadening the scope of non-stem cell-based applications in the area of secretomics.

Effect of serum on the differentiation of neural stem cells

To investigate the effect of serum on the differentiation of neural stem cells. The neural stem cells were isolated from the embryonic hippocampus tissues of Sprague Dawley rats at 14 day of pregnancy. After culturing and passaging, the 3rd generation cells were identified by immunocytochemical staining. Then, the cells were divided into 3 groups according to the concentrations of fetal bovine serum (FBS) used in the differentiation cell culture medium: 5% (group A), 1% (group B), 0 (group C), respectively. The other components of the culture media in 3 groups were the same. Cell viability was determined by using the Live/Dead cell staining at 8 days; the expressions of glial cell marker [glial fibrillary acidic protein (GFAP)] and neuronal marker (β-Ⅲ Tubulin) were determined and analyzed by immunocytochemical staining and real-time fluorescent PCR at 4 and 8 days of culture. Based on cell morphology and immunocytochemical staining, neural stem cells were identified. Cells were growing well with no death in all groups. With decreasing FBS concentration, the expression of GFAP was significantly decreased on both protein and mRNA level, whereas the expression of β-Ⅲ Tubulin was evidently increased. The staining of each group at 8 days was more obvious than that at 4 days. There were significant differences in mRNA expressions of GFAP and β-Ⅲ Tubulin at 4 and 8 days between groups ( P<0.05). Serum can promote the differentiation of neural stem cells into glial cells. At the same time, it inhibits the differentiation of neural stem cells into neurons, the lower the serum concentration, the smaller the effect.

Methods and significance of the combined detection of HER2 gene amplification and chemosensitivity in gastric cancer.

This study aims to investigate the significance of combined detection of HER2 gene amplification and chemosensitivity in gastric cancer. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and fluorescence reverse-transcription polymerase chain reaction (RT-PCR) were used to analyze the expression of HER2 protein, HER2 gene amplification and the mRNA expression of ERCC1, TUBB3 and TYMS genes in 135 cases of gastric carcinoma. The expression rate of HER2 protein was 39.3% (53/135). Among these positive cases, patients with HER2 protein (3+) accounted for 9.6% (13/135), patients with HER2 protein (2+) accounted for 13.3% (18/135), and patients with HER2 protein (1+) accounted for 16.3% (22/135). The amplification rate of the HER2 gene was 35.8% (19/53). In the detection of the mRNA expression of ERCC1, TUBB3 and TYMS, 45 patients had low and moderate single gene expression, 50 patients had low and moderate double gene expression, 22 patients had low and moderate mRNA expression for ERCC1, TUBB3 and TYMS, and 18 patients had no low and moderate expression. Among the 53 patients with HER2 protein expression and 22 patients with low and moderate mRNA expression of ERCC1, TUBB3 and TYMS, 12 patients received chemotherapy and trastuzumab. Follow-up results revealed that HER2 gene status was positively correlated with the therapeutic effect of the combined treatment in patients with low mRNA expression of ERCC1, TUBB3 and TYMS. Among these patients, five patients with extensive HER2 (3+), HER2 cluster-specific amplification, and low mRNA expression of ERCC1, TUBB3 and TYMS had a total survival of up to 19.1 months. The detection of HER2 in gastric cancer is highly heterogeneity, and the combined detection of HER2 protein expression, HER2 gene amplification and chemosensitivity can provide important reference markers for the benefit of antitumor drugs.

Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

Heterozygous missense mutations in human TUBB3 gene result in a spectrum of brain malformations associated with defects in axon guidance, neuronal migration and differentiation. However, the molecular mechanisms underlying mutation-related axon guidance abnormalities are unclear. Recent studies have shown that netrin-1, a canonical guidance cue, induced the interaction of TUBB3 with the netrin receptor deleted in colorectal cancer (DCC). Furthermore, TUBB3 is required for netrin-1-induced axon outgrowth, branching and pathfinding. Here, we provide evidence that TUBB3 mutations impair netrin/DCC signaling in the developing nervous system. The interaction of DCC with most TUBB3 mutants (eight out of twelve) is significantly reduced compared to the wild-type TUBB3. TUBB3 mutants R262C and A302V exhibit decreased subcellular colocalization with DCC in the growth cones of primary neurons. Netrin-1 increases the interaction of endogenous DCC with wild-type human TUBB3, but not R262C or A302V, in primary neurons. Netrin-1 also increases co-sedimentation of DCC with polymerized microtubules (MTs) in primary neurons expressing the wild-type TUBB3, but not R262C or A302V. Expression of either R262C or A302V not only suppresses netrin-1-induced neurite outgrowth, branching and attraction in vitro, but also causes defects in spinal cord commissural axon (CA) projection and pathfinding in ovo. Our study reveals that missense TUBB3 mutations specifically disrupt netrin/DCC-mediated attractive signaling.

Fetal Cerebrospinal Fluid Promotes Proliferation and Neural Differentiation of Stromal Mesenchymal Stem Cells Derived from Bone Marrow

ABSTRACT Embryonic cerebrospinal fluid (E-CSF) contains many neurotrophic and growth factors, acts as a growth medium for cortical progenitors, and can modulate proliferation and differentiation of neural stem cells. Mesenchymal stem cells (MSCs) are multipotential stem cells that can differentiate into several types of mesenchymal cells as well as nonmesenchymal cells, such as neural cells. In the present study, the effect of E-CSF on proliferation and neural differentiation of bone marrow mesenchymal stem cells (BM-MSCs) was investigated to test whether E-CSF is capable of driving these cells down the neuronal line. To verify the multipotential characteristics of BM-MSCs, the cells were analyzed for their osteogenic and adipogenic potential. Expression of the neural markers, MAP-2 and β-III tubulin, was determined by Immunocytochemistry. BM-MSCs differentiate into neuronal cell types when exposed to b-FGF. BMMSCs cells cultured in medium supplemented with CSF showed significantly elevated proliferation relative to control cells in media alone. E-CSF (E17-E19) supports viability and stimulates proliferation and, significantly, neurogenic differentiation of BM-MSCs. The data presented support an important role for CSF components, specifically neurotrophic factors, in stem cell survival, proliferation and neuronal differentiation. It is crucial to understand this control by CSF to ensure success in neural stem cell therapies.

Leucodistrofia hipomielinizante de tipo 6. Claves clínicas y de neuroimagen en la detección de un nuevo caso

Hypomyelinating leukodystrophy-6 is a rare and early onset neurodegenerative disease which entails a clinical pattern of pyramidal-extrapyramidal and cerebellar involvement and it comes with a neuroimaging consisting of hypomielination, cerebellar hypoplasia and specific abnormalities in basal ganglia, particularly the absence or nearly absence of putamen and the possible loss of caudate's volume. It is due to an alteration in tubulin and it is determined by mutations in heterocygosis in TUBB4A gene, showing complete penetrance. An 8-year-old child with history of delayed motor development, tremor, dysathria, ataxia, nystagmus, cognitive deficit and dystonia with pattern of hypomielination, vermis hypoplasia and absence of putamen. These findings, although distinctive, had been underestimated in previous evaluations and their detection determined the analyse and identification of a pathogenic variant in TUBB4A gene. Progressive deterioration leads the patient to total dependence or death in infancy or youth and there is no specific treatment capable of modifying its natural course.