A potential posttranscriptional regulator for p60-katanin: miR-124-3p.

Katanin is a microtubule severing protein belonging to the ATPase family and consists of two subunits; p60-katanin synthesized by the KATNA1 gene and p80-katanin synthesized by the KATNB1 gene. Microtubule severing is one of the mechanisms that allow the reorganization of microtubules depending on cellular needs. While this reorganization of microtubules is associated with mitosis in dividing cells, it primarily takes part in the formation of structures such as axons and dendrites in nondividing mature neurons. Therefore, it is extremely important in neuronal branching. p60 and p80 katanin subunits coexist in the cell. While p60-katanin is responsible for cutting microtubules with its ATPase function, p80-katanin is responsible for the regulation of p60-katanin and its localization in the centrosome. Although katanin has vital functions in the cell, there are no known posttranscriptional regulators of it. MicroRNAs (miRNAs) are a group of small noncoding ribonucleotides that have been found to have important roles in regulating gene expression posttranscriptionally. Despite being important in gene regulation, so far no microRNA has been experimentally associated with katanin regulation. In this study, the effects of miR-124-3p, which we detected as a result of bioinformatics analysis to have the potential to bind to the p60 katanin mRNA, were investigated. For this aim, in this study, SH-SY5Y neuroblastoma cells were transfected with pre-miR-124-3p mimics and pre-mir miRNA precursor as a negative control, and the effect of this transfection on p60-katanin expression was measured at both RNA and protein levels by quantitative real-time PCR (qRT-PCR) and western blotting, respectively. The results of this study showed for the first time that miR-124-3p, which was predicted to bind p60-katanin mRNA by bioinformatic analysis, may regulate the expression of the KATNA1 gene. The data obtained within the scope of this study will make important contributions in order to better understand the regulation of the expression of p60-katanin which as well will have an incontrovertible impact on the understanding of the importance of cytoskeletal reorganization in both mitotic and postmitotic cells.

HNF1A-MODY Mutations in Nuclear Localization Signal Impair HNF1A-Import Receptor KPNA6 Interactions.

Mutations in hepatocyte nuclear factor (HNF)1A gene cause the most common form of Maturity-onset diabetes of the young (MODY), a monogenic subtype of diabetes mellitus. Functional characterization of mutant proteins reveals that mutations may disrupt DNA binding capacity, transactivation ability and nuclear localization of HNF1A depending on the position of the mutation. Previously identified Arg271Trp and Ser345Tyr mutations in HNF1A were found to be defective in nuclear localization. Arg271 residue resides in a region similar to classical nuclear localization signal (NLS) motif, while Ser345 does not. Importin α family members recognize NLS motifs on cargo proteins and subsequently translocate them into nucleus. Here, we first investigated the nuclear localization mechanism of wild type HNF1A protein. For this purpose, we analyzed the interaction of HNF1A with three mouse homolog importin α proteins (KPNA2, KPNA4 and KPNA6) by co-immunoprecipitation assay and molecular docking simulation. Hereby, KPNA6 was identified as the main import receptor, which is responsible for the transport of HNF1A into the nucleus. Immunolocalization studies in mouse pancreatic cells (Min6) also confirmed the co-localization of HNF1A and KPNA6 in the cytoplasm. Secondly, the interaction between KPNA6 and mutant HNF1A proteins (Arg271Trp and Ser345Tyr) was assessed. Co-immunoprecipitation studies revealed a reduced interaction compared to wild type HNF1A. Our study demonstrated for the first time that HNF1A transcription factor is recognized and transported by importin/karyopherin import family, and mutations in NLS motifs may disrupt the interaction leading to nuclear localization abnormalities and MODY phenotype.

p60-katanin: a novel interacting partner for p53.

Katanin, one of the best-characterized microtubule (MT) severing proteins, is composed of two subunits: catalytic p60-katanin, and regulatory p80-katanin. p60-katanin triggers MT reorganization by severing them. MT reorganization is essential for both mitotic cells and post-mitotic neurons in numerous vital processes such as intracellular transport, mitosis, cellular differentiation and apoptosis. Due to the deleterious effect of continuous severing for cells, p60-katanin requires a strategic regulation. However, there are only a few known regulators of p60-katanin. p53 functions in similar cellular processes as katanin such as cell cycle, differentiation, and apoptosis depending on its interacting partners. Considering this similarity, in this study we investigated p53 as a potential regulatory candidate of p60-katanin, and examined their interaction. Co-immunoprecipitation analyses revealed that p60-katanin interacts with p53. We were able to locate a potential interaction site for the two proteins by deleting different candidate regions We showed for the first time that p53 and p60-katanin interact. This interaction appears to occur via p53's DNA binding domain and p60-katanin's C-terminal. This study will pave the way for future studies regarding the functional outcomes of this interaction which is vital for understanding the regulation of cellular events such as cell cycle, differentiation, and apoptosis in disease and in health.

Neuroprotective strategies against calpain-mediated neurodegeneration.

Calpains are calcium-dependent proteolytic enzymes that have deleterious effects on neurons upon their pathological over-activation. According to the results of numerous studies to date, there is no doubt that abnormal calpain activation triggers activation and progression of apoptotic processes in neurodegeneration, leading to neuronal death. Thus, it is very crucial to unravel all the aspects of calpain-mediated neurodegeneration in order to protect neurons through eliminating or at least minimizing its lethal effects. Protecting neurons against calpain-activated apoptosis basically requires developing effective, reliable, and most importantly, therapeutically applicable approaches to succeed. From this aspect, the most significant studies focusing on preventing calpain-mediated neurodegeneration include blocking the N-methyl-d-aspartate (NMDA)-type glutamate receptor activities, which are closely related to calpain activation; directly inhibiting calpain itself via intrinsic or synthetic calpain inhibitors, or inhibiting its downstream processes; and utilizing the neuroprotectant steroid hormone estrogen and its receptors. In this review, the most remarkable neuroprotective strategies for calpain-mediated neurodegeneration are categorized and summarized with respect to their advantages and disadvantages over one another, in terms of their efficiency and applicability as a therapeutic regimen in the treatment of neurodegenerative diseases.

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.

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.

SpeedyRINGO inhibits calpain-directed apoptosis in neurons.

The calcium-activated proteolytic enzyme calpain is one of the key proteins that can directly or indirectly drive neurons into apoptosis. The indirect way is through cyclin dependent kinase 5 (CDK5), a non-mitotic kinase, which is upregulated through calpain overactivation and followed by a subsequent increase in p53 and active caspase-3 levels under neurodegenerative conditions. The direct way is the upregulation of p53 by calpain itself, since p53 is a substrate for it. SpeedyRINGO is an atypical cell cycle regulator that has been shown to have protective effects in mitotic cells against apoptosis by inhibiting caspase-3 activation when p53 is present. Our aim was to reveal possible protective effects of SpeedyRINGO against calpain-induced caspase-3 activation in neurons which is crucial in terms of providing novel insights in preventing the caspase-3 activation cascade in neurodegeneration. For this reason, mRNA and protein levels were analyzed by qRT-PCR, western blotting, and immunofluorescence. We show that calpain overactivation leads to the upregulation of p53 and a subsequent increase in active caspase-3 level, indicating activation of apoptotic machinery in neurons. This calpain-directed caspase-3 activation upon upregulation of p53 is inhibited by the expression of SpeedyRINGO in rat hippocampal neurons. Therefore, SpeedyRINGO acts as a savior for neurons that are under apoptosis due to caspase-3 activation.

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.

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.

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.

The microtubule-severing proteins spastin and katanin participate differently in the formation of axonal branches.

Neurons express two different microtubule-severing proteins, namely P60-katanin and spastin. Here, we performed studies on cultured neurons to ascertain whether these two proteins participate differently in axonal branch formation. P60-katanin is more highly expressed in the neuron, but spastin is more concentrated at sites of branch formation. Overexpression of spastin dramatically enhances the formation of branches, whereas overexpression of P60-katanin does not. The excess spastin results in large numbers of short microtubules, whereas the excess P60-katanin results in short microtubules intermingled with longer microtubules. We hypothesized that these different microtubule-severing patterns may be due to the presence of molecules such as tau on the microtubules that more strongly shield them from being severed by P60-katanin than by spastin. Consistent with this hypothesis, we found that axons depleted of tau show a greater propensity to branch, and that this is true whether or not the axons are also depleted of spastin. We propose that there are two modes by which microtubule severing is orchestrated during axonal branch formation, one based on the local concentration of spastin at branch sites and the other based on local detachment from microtubules of molecules such as tau that regulate the severing properties of P60-katanin.