Arzu Karabay

Elk-1 interacts with neuronal microtubules and relocalizes to the nucleus upon phosphorylation.

ETS domain transcription factor Elk-1 has been primarily studied in the regulation of genes in response to mitogenic stimuli, however the presence of Elk-1 in axonal projections of largely post-mitotic adult hippocampal sections has been reported. This finding has initially led us to a basic question: how is Elk-1 anchored to neuronal projections? To that end, we have investigated the intracellular localization of Elk-1 and its biochemical interactions with neuronal microtubules in model systems. Our results showed co-localization of Elk-1 with microtubules in hippocampal cultures and SH-SY5Y neuroblastoma cell lines, and have further demonstrated that Elk-1 protein can biochemically interact with microtubules in vitro. Analysis of the protein sequence has indicated many putative microtubule binding domains, with the strongest binding prediction in amino acids 314-325, and our results show that Elk-1 can bind to microtubules through most of these regions, but no interaction was observed through the DNA binding domain, where no putative binding motifs were predicted. We further show that upon serum induction, most of the phospho-Elk-1 translocates to the nucleus, which is independent of translation. We propose that Elk-1 is anchored to neuronal microtubules in resting or unstimulated cells, and upon stimulation is phosphorylated, which relocalizes phospho-Elk-1 to the nucleus in neurons.

Protein kinase C activation causes neurite retraction via cyclinD1 and p60-katanin increase in rat hippocampal neurons

Neurons are differentiated postmitotic cells residing in G0 phase of the cell cycle and are unable to proceed through G1 phase, in which cyclinD1 needs to be up‐regulated for initiation. Yet, a growing body of evidence has shown that cell cycle re‐activation via cyclinD1 up‐regulation drives neurons into apoptosis. By contrast, there is also evidence demonstrating cell cycle proteins playing roles in neuronal differentiation. cyclinD1 has been shown to be differently regulated by protein kinase C alpha (PKC‐α) in various mitotic cells. Based on these different effects, we investigated the role of PKC‐α on cyclinD1 regulation in hippocampal neurons. Neurons were treated with PKC activator, PMA, and analysed for subcellular distributions of PKC‐α and cyclinD1. Remarkably, PMA treatment increased nuclear PKC‐α and cyclinD1, but not PKC‐ε in hippocampal neurons. Increases in nuclear PKC‐α and cyclinD1 were accompanied by microtubule re‐organisation via increases in tau and retinoblastoma protein phosphorylation levels. Increased p60‐katanin and p53 changed the neuronal morphology into neurons with shorter, but increased number of side branches. Since up‐regulation of cell cycle is associated with apoptosis in neurons, we also analysed changes in Bax, Bcl‐2 early and PARP (poly(ADP‐ribose)polymerase), caspase3 late apoptotic markers. However, we did not observe any indication of apoptosis. These data suggest that in addition to their previously known roles in mitotic cells on cell cycle regulation, PKC‐α and cyclinD1 seem to be important for differentiation, and nuclear PKC‐α and cyclinD1 interfere with differentiation by promoting microtubule re‐organisation through PKC signaling without triggering apoptosis.

Novel POC1A mutation in primordial dwarfism reveals new insights for centriole biogenesis

POC1A encodes a WD repeat protein localizing to centrioles and spindle poles and is associated with short stature, onychodysplasia, facial dysmorphism and hypotrichosis (SOFT) syndrome. These main features are related to the defect in cell proliferation of chondrocytes in growth plate. In the current study, we aimed at identifying the molecular basis of two patients with primordial dwarfism (PD) in a single family through utilization of whole-exome sequencing. A novel homozygous p.T120A missense mutation was detected in POC1A in both patients, a known causative gene of SOFT syndrome, and confirmed using Sanger sequencing. To test the pathogenicity of the detected mutation, primary fibroblast cultures obtained from the patients and a control individual were used. For evaluating the global gene expression profile of cells carrying p.T120A mutation in POC1A, we performed the gene expression array and compared their expression profiles to those of control fibroblast cells. The gene expression array analysis showed that 4800 transcript probes were significantly deregulated in cells with p.T120A mutation in comparison to the control. GO term association results showed that deregulated genes are mostly involved in the extracellular matrix and cytoskeleton. Furthermore, the p.T120A missense mutation in POC1A caused the formation of abnormal mitotic spindle structure, including supernumerary centrosomes, and changes in POC1A were accompanied by alterations in another centrosome-associated WD repeat protein p80-katanin. As a result, we identified a novel mutation in POC1A of patients with PD and showed that this mutation causes the formation of multiple numbers of centrioles and multipolar spindles with abnormal chromosome arrangement.

Pin1 inhibition activates cyclin D and produces neurodegenerative pathology

J. Neurochem. (2012) 120, 430–439.

Cell cycle markers have different expression and localization patterns in neuron-like PC12 cells and primary hippocampal neurons

Neuron-like PC12 cells are extensively used in place of neurons in published studies. Aim of this paper has been to compare mRNA and protein expressions of cell cycle markers; cyclinA, B, D, E; Cdk1, 2 and 4; and p27 in post-mitotic primary hippocampal neurons, mitotically active PC12 cells and NGF-differentiated post-mitotic PC12 cells. Contrary to PC12 cells, in neurons, the presence of all these markers was detected only at mRNA level; except for cyclinA, cyclinE and Cdk4, which were detectable also at protein levels. In both NGF-treated PC12 cells and neurons, cyclinE was localized only in the nucleus. In NGF-treated PC12 cells cyclinD and Cdk4 were localized in the nucleus while, in neurons cyclinD expression was not detectable; Cdk4 was localized in the cytoplasm. In neurons, cyclinA was nuclear, whereas in NGF-treated PC12 cells, it was localized in the cell body and along the processes. These results suggest that PC12 cells and primary neurons are different in terms of cell cycle protein expressions and localizations. Thus, it may not be very appropriate to use these cells as neuronal model system in order to understand neuronal physiological activities, upstream of where may lie cell cycle activation triggered events.

Katanin-p80 Gene Promoter Characterization and Regulation via Elk1

Katanin is an ATPase family member protein that participates in microtubule severing. It has heterodimeric structure consisting of 60 kDa (katanin-p60) and 80 kDa (katanin-p80) subunits encoded by KATNA1 and KATNB1 genes, respectively. Katanin-p60 has the enzymatic activity for microtubule severing, whereas katanin-p80 consists of multiple domains with different functions such as targeting katanin-p60 to the centrosome, augmenting microtubule severing by katanin-p60, and even suppressing microtubule severing. Despite the various important functions of katanin-p80, its transcriptional regulation has not been studied yet. Elk1 transcription factor has been shown to interact with microtubules and regulate the transcription of another microtubule severing protein, spastin. In spite of katanin’s importance, and structural and functional similarities to spastin, there is no study on the transcriptional regulation of katanin yet. In this study, we aimed to characterize KATNB1 promoter and analyze the effects of Elk1 on katanin-p80 expression. We identified a 518- bp TATA-less promoter including a critical CpG island and GC boxes as an optimal promoter, and sequential deletion of CpG island and the GC elements gradually decreased the KATNB1 promoter activity. In addition, we showed Elk1 binding on the KATNB1 promoter by EMSA. We found that Elk1 activated KATNB1 promoter, and increased both mRNA and protein levels of katanin-p80 in SH-SY5Y cells. On the other hand, KCl treatment increasing SUMOylation decreased KATNB1 promoter activity. Since microtubule severing is an important cellular mechanism of which malfunctions result in serious diseases such as spastic paraplegia, Alzheimer’s disease and cell cycle related disorders, identification of KATNB1 transcriptional regulation is crucial in understanding the coordination of microtubule severing activity by different proteins in the cells.

SPG4 gene promoter regulation via Elk1 transcription factor

J. Neurochem. (2011) 117, 724–734.

Okadaic acid–induced tau hyperphosphorylation and the downregulation of Pin1 expression in primary cortical neurons

Hyperphosphorylation of tau leading to neurofibrillary tangles (NFT) is one of the key pathological hallmarks in neurodegenerative disorders such as Alzheimer disease (AD). Peptidyl-prolyl cis-trans isomerase (Pin1) regulates the phosphorylation of Ser/Thr sites of tau protein, and promotes microtubule assembly. In this study, we aimed to determine the effect of tau hyperphosphorylation on Pin1 expression in primary cortical neurons in order to investigate the results of the pathological process on Pin1, an important enzyme involved in various cellular mechanisms. Primary cortical neurons were prepared from embryonic day 16 -Sprague Dawley rat embryos. The cultures were treated with 25 nM okadaic acid (OKA) on day 7 in order to promote tau hyperphosphorylation. The cytotoxicity was determined with LDH release and measured by ELISA. Tau phosphorylation was confirmed by western blot using anti-tau antibodies Thr231 and Tau-1. Pin1 mRNA expression level was determined by qRT-PCR at 8 and 24 h. Pin1 protein expression was analyzed with immunofluorescent labeling at 8 and 24 h. Tau phosphorylation on Thr231 was increased and non-phosphorylated Tau-1 was decreased in OKA treated group compared with the untreated control at 8 h of treatment. While Pin1 mRNA expression levels at 8 h post-OKA treatment were lower than that of control groups, there were no differences between OKA-treated group and control groups in Pin1 protein expression. Whereas no significant differences for Pin1 mRNA expression, protein expression levels were decreased OKA-treated group compared to control groups at 24 h of treatment. The LDH release of OKA-treated group was significantly increased at 24 h. Our study indicates that although OKA treatment suppressed Pin1 mRNA expression and induced tau phosphorylation at 8 h of treatment, its influence on Pin1 protein expression has 16 h phase delay. Given the important role of Pin1 in many cellular mechanisms these results might indicate that tau hyperphosphorylation involved in many neurodegenerative disorders may cause some alterations in brain microenvironment via Pin1.This is the first demonstration of the alteration of the Pin1 mRNA and protein expression in OKA induced model in primary cortical neurons.

Expression of cell cycle proteins in cortical neurons-Correlation with glutamate-induced neurotoxicity.

Under physiological conditions, upon differentiation neurons become irreversibly post-mitotic by down-regulating cell cycle progression. However, recent studies have provided evidence that aberrant expression of cell cycle related proteins; especially cyclins, cyclin-dependent kinases, and their inhibitors are accompanied by programmed cell death in neurons. This abnormal phenotype has been postulated to contribute to the pathophysiology of different neurodegenerative diseases. Glutamate is the most abundant and major excitatory neurotransmitter in the central nervous system but high concentrations are reported to be involved in the pathology of many neurodegenerative diseases. The mechanisms of glutamate neurotoxicity have been intensively investigated over the past decades but still remain not fully understood. In this study, we hypothesized that aberrant regulation of cell cycle proteins may be involved in glutamate-induced neurotoxicity in primary cultures of rat cortical neurons. The results have shown that, glutamate treatment caused apoptosis by inducing active caspase-3 and p53 expression. Together with this, an increase in cyclin D1 and Cdk4 protein levels, localization of cyclin D1 to nucleus, and a decrease in the cell cycle inhibitor p27 were observed. After glutamate treatment we also detected up-regulation of protein kinase C-α (PKC-α) protein expression. Altogether, the data reported in this study show for the first time that glutamate in cortical neurons changes simultaneously the expression levels of a number of key cell cycle proteins and cell homeostasis regulators.

IGF-1 Participates Differently in Regulation of Severing Activity of Katanin and Spastin

Spastin and p60-katanin are AAA family proteins that participate in microtubule severing, while lipotransin, another AAA family protein is a hormone sensitive lipase interacting protein. Sequence alignment analysis suggests that lipotransin and human p60-katanin are the orthologs of each other. Studies identified that insulin may negatively regulate ATPase activity of lipotransin. To reveal the effects of insulin on regulation of severing activity of p60-katanin and spastin, hippocampal neurons over-expressing spastin and p60-katanin were treated with IGF-1. Changes in neuronal branching by considering the total process lengths and average process numbers were quantitatively analyzed. According to the results of this study, total process lengths of hippocampal neurons and average process numbers remained similar in control and p60-katanin over-expressing neurons upon IGF-1 treatment, while significant decrease was observed in spastin over-expressing neurons. This study indicated that IGF-1 participates differently in the regulation of spastin and p60-katanin in terms of neuronal branching.