Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line

Summary. No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.     

Aberrant expression of cytoskeleton proteins in hippocampus from patients with mesial temporal lobe epilepsy

Summary. Mesial temporal lobe epilepsy (MTLE), the most common form of epilepsy, is characterised by cytoarchitectural abnormalities including neuronal cell loss and reactive gliosis in hippocampus. Determination of aberrant cytoskeleton protein expression by proteomics techniques may help to understand pathomechanism that is still elusive. We searched for differential expression of hippocampal proteins by an analytical method based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry unambiguously identifying 77 proteins analysed in eight control and eight MTLE hippocampi. Proteins were quantified and we observed 18 proteins that were altered in MTLE. Cytoskeleton proteins tubulin α-1 chain, β-tubulin, profilin II, neuronal tropomodulin were significantly reduced and one actin spot was missing, whereas ezrin and vinculin were significantly increased in MTLE. Proteins of several classes as e.g. antioxidant proteins (peroxiredoxins 3 and 6), chaperons (T-complex protein 1-α, stress-induced-phosphoprotein 1), signaling protein MAP kinase kinase 1, synaptosomal proteins (synaptotagmin I, α-synuclein), NAD-dependent deacetylase sirtuin-2 and 26S protease regulatory subunit 7 protein, neuronal-specific septin 3 were altered in MTLE. Taken together, the findings may represent or lead to cytoskeletal impairment; aberrant antioxidant proteins, chaperons, MAP kinase kinase 1 and NAD-dependent deacetylase sirtuin-2 may have been involved in pathogenetic mechanisms and altered synaptosomal protein expression possibly reflects synaptic impairment in MTLE. J. W. Yang and T. Czech have equally contributed to the paper.     

The oxidant defense system in human neuroblastoma IMR-32 cells predifferentiation and postdifferentiation to neuronal phenotypes

Differentiated neurons were investigated for their susceptibility to oxidative damage based on variations in the oxidant defense system occurring during differentiation. The main antioxidant enzymes and substances in human neuroblastoma (IMR-32) cells were evaluated pre- and post-differentiation to a neuronal phenotype. The activity of CuZn superoxide dismutase (CuZnSOD) and Mn superoxide dismutase (MnSOD) and the concentration of CuZnSOD were higher, but the activity and concentration of catalase were lower after differentiation. Differentiated cells had higher activity of glutathione peroxidase (GPx), lower concentration of total glutathione, a higher ratio of oxidised/reduced glutathione and lower activity of glucose-6-phosphate dehydrogenase than undifferentiated cells. We conclude that differentiated neuronal cells may be highly susceptible to oxidant-mediated damage based on the relative activities of the main antioxidant enzymes and on a limited capacity to synthesise and/or recycle glutathione.     

Nectin-like molecule 1 is a high abundance protein in cerebellar neurons

Summary. Nectins and Nectin-like molecules belong to the Ca-independent immunoglobulin superfamily of cell adhesion molecules and are mandatory for various cellular functions such as morphogenesis, differentiation and proliferation. Among them, Nectin-like molecule 1 (Necl-1) is unique for its exclusive expression in the brain where it is localized at the contact sites among axon terminals and glia cell processes, cooperatively forming synapses. We hereby aimed to unambiguously characterize Necl-1 at the protein level in rat brain. Rat cerebellar neurons were lysed, proteins extracted and run on two-dimensional gel electrophoresis with subsequent in-gel digestion and mass spectrometrical (MS/MS) analysis of protein spots. One spot at pI 5.96 with an observed molecular weight of 26 kDa was identified as Nectin-like molecule 1. MS/MS analyses of three matching peptides warranted unambiguous identification for the first time. Additionally, we verified the result by immunoblotting and detected two bands at about 48 kDa and 60 kDa. The proposed roles of Necl-1 in cerebellar morphogenesis as well as plasticity of synapses challenge further research on its function in more detail and we hereby provide a fair analytical tool for the unequivocal determination of Necl-1, independent of antibody availability and specificity.     

Antioxidant defense and apoptotic effectors in ascorbic acid and β-glycerophosphate-induced osteoblastic differentiation

MC3T3-E1 cells grown in the presence of ascorbic acid and β-glycerophosphate (AA/β-GP) express alkaline phosphatase and produce an extensive collagenous extracellular matrix. Differentiated MC3T3-E1 cells are more sensitive to hydrogen peroxide-induced oxidative stress than undifferentiated cells. In this study, we compared the profile of antioxidant enzymes and molecular markers of apoptosis in undifferentiated and differentiated MC3T3-E1 cells (cell differentiation was induced by treatment with AA/β-GP). Differentiated osteoblasts showed lower expression and activity of catalase, glutathione S-transferase and glutathione peroxidase. The total superoxide dismutase activity and the expression of Cu/Zn superoxide dismutase were also lower, while the expression of Mn superoxide dismutase was higher in differentiated osteoblasts. The level of malondialdehyde, a widely used marker for oxidative stress, was lower in the AA/β-GP group compared with control cells, but this difference was not significant. Western blotting showed that treatment with AA/β-GP increased the Bax/Bcl-2 ratio used as an index of cellular vulnerability to apoptosis. In addition, the activities of caspases 3, 8 and 9 and cleaved poly (ADP) ribose polymerase were significantly higher in differentiated cells. These findings provide new insights into how changes in the activities of major antioxidant enzymes and in the signaling pathways associated with apoptosis may influence the susceptibility of bone cells to oxidative stress.     

Proteome analysis of primary neurons and astrocytes from rat cerebellum

Neurons and astrocytes are predominant cell types in brain and have distinguished morphological and functional features. Although several proteomics studies were carried out on the brain, work on individual brain cells is limited. Generating individual proteomes of neurons and astrocytes, however, is mandatory to assign protein expression to cell types rather than to tissues. We aimed to provide maps of rat primary neurons and astrocytes using two-dimensional gel electrophoresis with subsequent in-gel digestion, followed by MALDI-TOF/TOF. 428 protein spots corresponding to 226 individual proteins in neurons and 406 protein spots representing 228 proteins in astrocytes were unambiguously identified. Proteome data include proteins from several cascades differentially expressed in neurons and astrocytes, and specific expressional patterns of antioxidant, signaling, chaperone, cytoskeleton, nucleic acid binding, proteasomal, and metabolic proteins are demonstrated. We herein present a reference database of primary rat primary neuron and astrocyte proteomes and provide an analytical tool for these structures. The concomitant expressional patterns of several protein classes are given and potential neuronal and astrocytic marker candidates are presented.     

Differentiation-dependent expression of hypothetical proteins in the neuroblastoma cell line N1E-115

Several protein cascades, including signaling, cytoskeletal, chaperones, metabolic, and antioxidant proteins, have been shown to be involved in the process of neuronal differentiation (ND) of neuroblastoma cell lines. No systematic approach to detect hitherto unknown and unnamed proteins or structures that have been predicted upon nucleic acid sequences in ND has been published so far. We therefore decided to screen hypothetical protein (HP) expression by protein profiling. Two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) identification was used for expression analysis of undifferentiated and dimethylsulfoxide-induced neuronally differentiated N1E-115 cells. We unambiguously identified six HPs: Q8C520, Q99LF4, Q9CXS1, Q9DAF8, Q91WT0, and Q8C5G2. A prefoldin domain in Q91WT0, a t-SNARE domain in Q9CXS1, and a bromodomain were observed in Q8C5G2. For the three remaining proteins, no putative function using Pfam, BLOCKS, PROSITE, PRINTS, InterPro, Superfamily, CoPS, and ExPASy could be assigned. While two proteins were present in both cell lines, Q9CXS1 was switched off (i.e., undetectably low) in differentiated cells only, and Q9DAF8, Q91WT0, and Q8C5G2 were switched on in differentiated cells exclusively. Herein, using a proteomic approach suitable for screening and identification of HP, we present HP structures that have been only predicted so far based upon nucleic acid sequences. The four differentially regulated HPs may play a putative role in the process of ND.     

Predicting secretory protein signal sequence cleavage sites by fusing the marks of global alignments

Summary. A newly synthesized secretory protein in cells bears a special sequence, called signal peptide or sequence, which plays the role of “address tag” in guiding the protein to wherever it is needed. Such a unique function of signal sequences has stimulated novel strategies for drug design or reprogramming cells for gene therapy. To realize these new ideas and plans, however, it is important to develop an automated method for fast and accurately identifying the signal sequences or their cleavage sites. In this paper, a new method is developed for predicting the signal sequence of a query secretory protein by fusing the results from a series of global alignments through a voting system. The very high success rates thus obtained suggest that the novel approach is very promising, and that the new method may become a useful vehicle in identifying signal sequence, or at least serve as a complementary tool to the existing algorithms of this field.     

Proteome analysis of mouse primary astrocytes

Astrocytes play a role in energy metabolism, neuronal homeostasis and release of neuronal growth factors and several neurotransmitters. They also relate to a variety of brain diseases and contribute to restore brain dysfunction. Although current research has revealed several roles for astrocytes, knowledge on astrocytic protein expression is limited and a systematic and comprehensive proteome study of astrocytes has not been reported so far. We applied a proteomics technique based on two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF) and unambiguously identified 301 spots corresponding to 191 individual proteins in primary mouse astrocytes. The identified proteins were from antioxidant, chaperone, cytoskeleton, nucleic acid binding, signaling, proteasomal, hypothetical and miscellaneous proteins. A reference database is provided and proteins were identified in astrocytes specifically and unambiguously for the first time. A reliable analytical tool independent of antibody availability and specificity along with tentative astrocytic marker proteins is described.     

Neuronal Differentiation of LUHMES Cells Induces Substantial Changes of the Proteome

Neuronal cell lines are important model systems to study mechanisms of neurodegenerative diseases. One example is the Lund Human Mesencephalic (LUHMES) cell line, which can differentiate into dopaminergic‐like neurons and is frequently used to study mechanisms of Parkinson's disease and neurotoxicity. Neuronal differentiation of LUHMES cells is commonly verified with selected neuronal markers, but little is known about the proteome‐wide protein abundance changes during differentiation. Using mass spectrometry and label‐free quantification (LFQ), the proteome of differentiated and undifferentiated LUHMES cells and of primary murine midbrain neurons are compared. Neuronal differentiation induced substantial changes of the LUHMES cell proteome, with proliferation‐related proteins being strongly down‐regulated and neuronal and dopaminergic proteins, such as L1CAM and α‐synuclein (SNCA) being up to 1,000‐fold up‐regulated. Several of these proteins, including MAPT and SYN1, may be useful as new markers for experimentally validating neuronal differentiation of LUHMES cells. Primary midbrain neurons are slightly more closely related to differentiated than to undifferentiated LUHMES cells, in particular with respect to the abundance of proteins related to neurodegeneration. In summary, the analysis demonstrates that differentiated LUHMES cells are a suitable model for studies on neurodegeneration and provides a resource of the proteome‐wide changes during neuronal differentiation. (ProteomeXchange identifier PXD020044).     

Planarian pharynx regeneration in regenerating tail fragments monitored with cell-specific monoclonal antibodies

 The special morphological features of freshwater planarians make them an attractive and informative model for studying the processes of regeneration and pattern formation. In this work, we investigate pattern formation and maturation of the planarian pharynx during regeneration in tail fragments. Using three monoclonal antibodies (TCAV-1, TF-26 and TMUS-13) specific for epithelial, secretory and muscle cells, respectively, we followed the sequence and timing of differentiation and maturation of these three cell types within the regenerating pharynx. Two of these monoclonal antibodies, TCAV-1 and TMUS-13, also labelled morphologically immature cells that appear to be committed to the differentiation pathway leading to their respective adult cell types. Our results show that the cells forming the new pharynx come from undifferentiated cells through proliferation and differentiation processes rather than from differentiated cells of the old stump. We describe three stages of pharynx regeneration according to the immunoreactivity shown: (1) no immunoreactivity, corresponding to the accumulation of undifferentiated cells that form the pharynx primordium; (2) immunoreactivity to TCAV-1 and TMUS-13, corresponding to the re-building of the pharynx; and (3) immunoreactivity to TF-26, corresponding to a fully mature and functional pharynx. The sequence of differentiation of these three cell types suggests that the pharynx grows by intercalation of new undifferentiated cells coming from the parenchyma between the older pharyngeal cells, in agreement with existing models of pharynx regeneration. Finally, our results suggest an intercalary model for pharynx epithelial cell renewal. Received: 30 September 1996 / Accepted: 6 December 1996     

Mass spectrometrical analysis of phosphoprotein enriched in astrocytes of 15 kDa in mouse hippocampi

Summary. Phosphoprotein enriched in astrocytes of 15kDa (PEA-15) is a small protein that was first identified as an abundant phosphoprotein in brain. PEA-15 was characterised so far at the immunochemical level and by a microsequencing attempt. In order to update characterisation of this important structure by advanced methodology unambiguously identifying proteins independent of antibody availability and specificity, we used a proteomic method for this purpose: Performing protein profiling in mouse hippocampi using two dimensional gel electrophoresis with subsequent mass spectrometrical (MS/MS) identification we detected this protein and demonstrate proteomic characterisation of PEA-15 (Q62048). This study enables further specific and unambiguous determination serving as an analytical tool.     

Expression of Calmodulin-Dependent Phosphodiesterase, Calmodulin-Dependent Protein Phosphatase, and Other Calmodulin-Binding Proteins in Human SMS-KCNR Neuroblastoma Cells

Calmodulin (CaM)-dependent enzymes, such as CaM-dependent phosphodiesterase (CaM-PDE), CaM-dependent protein phosphatase (CN), and CaM-dependent protein kinase II (CaM kinase II), are found in high concentrations in differentiated mammalian neurons. In order to determine whether neuroblastoma cells express these CaM-dependent enzymes as a consequence of cellular differentiation, a series of experiments was performed on human SMS-KCNR neuroblastoma cells; these cells morphologically differentiate in response to retinoic acid and phorbol esters [12-O-tetradecanoylphorbol 13-acetate (TPA)]. Using biotinylated CaM overlay procedures, immunoblotting, and protein phosphorylation assays, we found that SMS-KCNR cells expressed CN and CaM-PDE, but did not appear to have other neuronal CaM-binding proteins. Exposure to retinoic acid, TPA, or conditioned media from human HTB-14 glioma cells did not markedly alter the expression of CaM-binding proteins; 21-day treatment with retinoic acid, however, did induce expression of novel CaM-binding proteins of 74 and 76 kilodaltons. Using affinity-purified polyclonal antibodies, CaM-PDE immunoreactivity was detected as a 75-kilodalton peptide in undifferentiated cells, but as a 61-kilodalton peptide in differentiated cells. CaM kinase II activity and subunit autophosphorylation was not evident in either undifferentiated or neurite-bearing cells; however, CaM-dependent phosphatase activity was seen. Immunoblot analysis with affinity-purified antibodies against CN indicated that this enzyme was present in SMS-KCNR cells regardless of their state of differentiation. Although SMS-KCNR cells did not show a complete pattern of neuronal CaM-binding proteins, particularly because CaM kinase II activity was lacking, they may be useful models for examination of CaM-PDE and CN expression. It is possible that CaM-dependent enzymes can be used as sensitive markers for terminal neuronal differentiation.     

Induction of apoptosis and differentiation in neuroblastoma and astrocytoma cells by the overexpression of Bin1, a novel Myc interacting protein.

Bin1 is a novel protein that specifically binds Myc and inhibits, at least in part, Myc transactivation. Bin1 seems to play a role in cell cycle control, acting as a tumor suppressor gene. Since MYC family genes play a regulatory role in the proliferation, differentiation, and apoptosis of the nervous system, we studied the effects of the overexpression of the Myc-interacting protein, Bin1, in neuroblastoma and astrocytoma cell lines, which were chosen as neural cell system models. The major effects of BIN1 overexpression observed in undifferentiated neuroblastoma and astrocytoma cells were a significant reduction of cell growth, an increase in the G(0)/G(1) cell population and the induction of apoptosis. The trigger of programmed cell death by Bin1 is described for the first time. Bin1 overexpression in undifferentiated cells did not induce any maturation process as neither neuronal nor astrocyte differentiation markers were upregulated in neuroblastoma and astrocytoma cells, respectively. On the other side, the effects of Bin1 overproduction in neuroblastoma and astrocytoma cells committed towards neuronal and astrocyte differentiation, respectively, were different from those observed in undifferentiated cells. Although we did not evidence any triggering of programmed cell death, we did notice a further induction towards more differentiated phenotypes. Our studies suggest that Bin1 overexpression in neuroblastoma and astrocytoma cells can result in one of the following pathways: (1) suppressed cell proliferation, (2) induced differentiation, or (3) apoptosis. Thus, it appears that Bin1 operates through different pathways that involve activation of different genes: the chosen pathway however will depend on the proliferating or differentiated state of the cell.     

Role of reactive oxygen species in extracellular signal-regulated protein kinase phosphorylation and 6-hydroxydopamine cytotoxicity

A number of reports indicate the potential for redox signalling via extracellular signal-regulated protein kinases (ERK) during neuronal injury. We have previously found that sustained ERK activation contributes to toxicity elicited by 6-hydroxydopamine (6-OHDA) in the B65 neuronal cell line. To determine whether reactive oxygen species (ROS) play a role in mediating ERK activation and 6-OHDA toxicity, we examined the effects of catalase, superoxide dismutase (SOD1), and metalloporphyrin antioxidants ('SOD mimetics') on 6-OHDA-treated cells. We found that catalase and metalloporphyrin antioxidants not only conferred protection against 6-OHDA but also inhibited development of sustained ERK phosphorylation in both differentiated and undifferentiated B65 cells. However, exogenously added SOD1 and heat-inactivated catalase had no effect on either toxicity or sustained ERK phosphorylation. This correlation between antioxidant protection and inhibition of 6-OHDA-induced sustained ERK phosphorylation suggests that redox regulation of ERK signalling cascades may contribute to neuronal toxicity.     

Involvement of caveolin-1 in cholesterol enrichment of high density lipoprotein during its assembly by apolipoprotein and THP-1 cells

High density lipoprotein (HDL) is assembled by interaction of apolipoprotein A-I with human monocytic leukemia cell line THP-1 by removing cellular cholesterol and phospholipid. Although the HDL formed with undifferentiated THP-1 cells contained only phosphatidylcholine and almost no cholesterol, the cells differentiated with phorbol 12-myristate 13-acetate (PMA) generated HDL enriched in cholesterol. The extent of cholesterol enrichment related to the cellular cholesterol level in the differentiated cells, but only weakly in the undifferentiated cells. In contrast, the differentiation had no influence on the diffusion-mediated cellular cholesterol efflux. The undifferentiated cells expressed the messages of ATP-binding cassette transporter 1 and caveolin-1, at low levels, and the PMA-induced differentiation resulted in substantial expression of both messages. Caveolin-1 protein expression was also highly induced by the PMA treatment of THP-1 cells. When the cells were treated with the antisense DNA of caveolin-1 and differentiated, both caveolin-1 synthesis and cholesterol incorporation into the HDL were reduced in parallel to generate the cholesterol-poor HDL.We concluded that caveolin-1 is involved in enrichment with cholesterol of the HDL generated by the apolipoprotein-cell interaction. This function is independent of the assembly of HDL particles with cellular phospholipid and of nonspecific, diffusion-mediated efflux of cellular cholesterol.