Microtubule-Associated Protein 2 as a DHEA Binding Protein in Endometrial Cancer

Dehydroepiandrosterone (DHEA) is an androgen secreted by the adrenal glands, but its binding affinity for the androgen receptor is very low. DHEA is transformed into androstenedione by 3β-hydroxysteroid dehydrogenase (HSD) and then into testosterone by 17β-HSD type 5, or into estrone by aromatase. DHEA is also converted into androstenediol by 17β-HSD type 1. Therefore, DHEA is considered to play an important role as a precursor hormone for sex steroid hormones. We performed a search for a protein having an amino acid sequence homology to the DHEA binding site of 17β-HSD type 1, and found that microtubule-associated protein 2 (MAP2) binds to DHEA (Laurine E et al., J Biol Chem. 2003). MAP2 expression is necessary for neurite extension and cessation of cell division. MAP2 is known to suppress migration and invasion and affect the assembly, stabilization, and bundling of microtubules in melanoma cells, but the function of MAP2 in endometrial cancer has not been clarified. In this study, we investigated the expression of MAP2 and its association with DHEA in order to clarify the direct non-receptor action of DHEA in endometrial cancer. We employed frozen and formalin-fixed paraffin-embedded (FFPE) tissues of 35 endometrial cancer tissues (G1, n=12; G2, n=10; G3, n=9; Serous, n=4). Hormone concentrations were measured by liquid chromatograph-tandem mass spectrometer from the frozen sample, and immunohistochemistry of MAP2 was performed using FFPE tissues. We also examined MAP2 immunoreactivity using 59 normal endometrial tissues (proliferative phase, n=33; secretory phase, n=26) of FFPE tissue microarray slides. MAP2 immunoreactivity was found in the cytoplasm of endometrial cancer cells, and the MAP2-positive rate was significantly higher in type 1 (G1 and G2) than in type 2 (G2 and G3). The cell proliferation marker Ki-67 index was significantly lower in the MAP2-positive group. MAP2 was also detected in the glandular epithelial cells of the normal endometrium. The MAP2-positive rate was lower in the proliferative phase than in the secretory phase. Furthermore, the concentration of DHEA in the cancer tissue was significantly higher in the MAP2-positive group than in the MAP2-negative group. MAP2 is known to act on the stability of microtubules and is thought to be involved in the suppression of proliferation and infiltration in cancer cells. It was suggested that DHEA is involved in the stabilization of MAP2 and suppresses the progression of cancer in a hormone receptor-independent manner.

Genetic and microstructural differences in the cortical plate of gyri and sulci during gyrification in fetal sheep

Gyrification of the cerebral cortex is a developmentally important process, but the mechanisms that drive cortical folding are not fully known. Theories propose that changes within the cortical plate (CP) cause gyrification, yet differences between the CP below gyri and sulci have not been investigated. Here we report genetic and microstructural differences in the CP below gyri and sulci assessed before (at 70 days of gestational age [GA] 70), during (GA 90), and after (GA 110) gyrification in fetal sheep. The areal density of BDNF, CDK5, and NeuroD6 immunopositive cells were increased, and HDAC5 and MeCP2 mRNA levels were decreased in the CP below gyri compared with sulci during gyrification, but not before. Only the areal density of BDNF-immunopositive cells remained increased after gyrification. MAP2 immunoreactivity and neurite outgrowth were also increased in the CP below gyri compared with sulci at GA 90, and this was associated with microstructural changes assessed via diffusion tensor imaging and neurite orientation dispersion and density imaging at GA 98. Differential neurite outgrowth may therefore explain the localized changes in CP architecture that result in gyrification.

CDK5 Knockdown Prevents Hippocampal Degeneration and Cognitive Dysfunction Produced by Cerebral Ischemia

Acute ischemic stroke is a cerebrovascular accident and it is the most common cause of physical disabilities around the globe. Patients may present with repeated ictuses, experiencing mental consequences, such as depression and cognitive disorders. Cyclin-dependent kinase 5 (CDK5) is a kinase that is involved in neurotransmission and plasticity, but its dysregulation contributes to cognitive disorders and dementia. Gene therapy targeting CDK5 was administered to the right hippocampus of ischemic rats during transient cerebral middle artery occlusion. Physiologic parameters (blood pressure, pH, pO2, and pCO2) were measured. The CDK5 downregulation resulted in neurologic and motor improvement during the first week after ischemia. Cyclin-dependent kinase 5 RNA interference (RNAi) prevented dysfunctions in learning, memory, and reversal learning at 1 month after ischemia. These observations were supported by the prevention of neuronal loss, the reduction of microtubule-associated protein 2 (MAP2) immunoreactivity, and a decrease in astroglial and microglia hyperreactivities and tauopathy. Additionally, CDK5 silencing led to an increase in the expression of brain-derived neurotrophic factor (BDNF), its Tropomyosin Receptor kinase B (TRKB) receptor, and activation of cyclic AMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK), which are important targets in neuronal plasticity. Together, our findings suggest that gene therapy based on CDK5 silencing prevents cerebral ischemia-induced neurodegeneration and motor and cognitive deficits.

Changes in Extracellular Action Potential Detect Kainic Acid and Trimethyltin Toxicity in Hippocampal Slice Preparations Earlier than do MAP2 Density Measurements

In vitro electrophysiological techniques for the assessment of neurotoxicity could have several advantages over other methods in current use, including the ability to detect damage at a very early stage, and could further assist in replacing animal experimentation in vivo. We investigated how an electrophysiological parameter, the extracellularly-recorded compound action potential (“population spike”, PS) could be used as a marker of in vitro neurotoxicity in the case of two well-known toxic compounds, kainic acid (KA) and trimethyltin (TMT). We compared the use of this electrophysiological endpoint with changes in immunoreactivity for microtubule-associated protein 2 (MAP2), a standard histological test for neurotoxicity. We found that both toxic compounds reliably caused disappearance of the PS, and that such disappearance occurred after only 1 hour of exposure to the drug. By contrast, densitometric measurements of MAP2 immunoreactivity were unaffected by both KA and TMT after such a short exposure time. We conclude that, in the case of KA and TMT, the extracellular PS was abolished at a very early time-point, when MAP2 immunoreactivity levels were still comparable to those of the untreated controls. Electrophysiology could be a reliable and early indicator of neurotoxicity, which could improve our ability to test for neurotoxicity in vitro, thus further replacing the need for in vivo experimentation.

Time Course and Cellular Distribution of Hsp27 and Hsp72 Stress Protein Expression in a Quantitative Gerbil Model of Ischemic Injury and Tolerance: Thresholds for Hsp72 Induction and Hilar Lesioning in the Context of Ischemic Preconditioning

The distribution and time course of expression of the heat shock/stress proteins, hsp27 and hsp72, were evaluated in a highly controlled gerbil model of ischemic injury and tolerance induction, in which the duration of ischemic depolarization in each hippocampus provides a precise quantitative index of insult severity. Gerbils were subjected to brief priming insults (2- to 3.5-minute depolarization) that produce optimal preconditioning, to severe test insults (6- to 8.5-minute depolarization) that produce complete CA1 neuron loss in naive animals, or to combined insults administered 1 week apart, after which almost complete tolerance to CA1 neuron injury is observed. Immunoreactivities of hsp27, hsp72, glial fibrillary acidic protein and microtubule-associated protein 2 (MAP2) were evaluated in animals perfused at defined intervals after the final insult in each treatment group, using a variation of established antigen-retrieval procedures that significantly improves detection of many proteins in vibratome brain sections. Hsp72 was detected in CA1 neurons of some hippocampi 2 to 4 days after preconditioning, but this was only seen after the longest priming depolarizations, whereas shorter insults that still induced optimal tolerance failed to induce hsp72. Hsp72 was induced after test insults in preconditioned hippocampi, but at a higher depolarization threshold than observed for naive animals. An astrocytic localization of hsp27 was observed in regions of neuron injury, as indicated by reduced MAP2 immunoreactivity, and was primarily restricted to dentate hilus after preconditioning insults. These results establish that limited hilar lesions are characteristic of optimal preconditioning, whereas prior neuronal expression of either hsp72 or hsp27 is not required for ischemic tolerance.

Transcortical Alterations inNa+-K+ATPase and Microtubule-Associated Proteins Immunoreactivity in the Rat Cortical Atrophy Model Induced by Hypoxic Ischemia

To identify the chronological transcortical change in the contralateral hemisphere following ischemic insults, we investigated the changes in microtubule associated protein (MAP) andNa+-K+ATPase expressions in the peri-infarct zone and contralateral hemisphere, including the hippocampus. Two days after hypoxic ischemia,Na+-K+ATPase immunoreactivity was significantly enhanced in the contralateral cortex and was maintained up to 7 days after ischemia, whereasNa+-K+ATPase immunoreactivity in the peri- and infarct zones was unaffected by hypoxic ischemia. In contrast, 2 to 7 days after ischemia, MAP1A and MAP2 immunoreactivity in the ipsi- and contralateral cortex significantly decreased, whereas in layer V, MAP1 immunoreactivity obviously accumulated in the neurons and their processes. In the hippocampus, 2 days after insults both MAP1A and MAP2 immunoreactivity was significantly reduced within the ipsi- and contralateral hippocampus. In the contralateral hippocampus, however, the distribution of MAP2 immunoreactivity recovered to the sham level 7 days after ischemia, whereas MAP1A immunoreactive axons remained 2 months after ischemia. The results suggest that the unilateral elevation ofNa+-K+ATPase immunoreactivity reflects elevated neuronal activity. In addition, this asymmetric hyperexcitability might play an important role in the recovery or the reorganization of the brain, accompanied by transcortical changes in MAPs expression.