17ß-Estradiol inhibits apoptotic cell death of oligodendrocytes by inhibiting RhoA-JNK3 activation after spinal cord injury.

A delayed oligodendrocyte cell death after spinal cord injury (SCI) contributes to chronic demyelination of spared axons, leading to a permanent neurological deficit. Therefore, therapeutic approaches to prevent oligodendrocyte cell death after SCI should be considered. Estrogens are well known to have a broad neuroprotective effect, but the protective effect of estrogens on oligodendrocytes after injury is largely unknown. Here, we demonstrated that 17ß-estradiol attenuates apoptosis of oligodendrocytes by inhibiting RhoA and c-Jun-N-terminal kinase activation after SCI. Estrogen receptor (ER)-α and -ß were expressed in oligodendrocytes of the spinal cord, and 17ß-estradiol treatment significantly inhibited oligodendrocyte cell death at 7 d after injury as compared with vehicle (cyclodextrin) control. 17ß-Estradiol also attenuated caspase-3 and -9 activation at 7 d and reduced the loss of axons from progressive degeneration. In addition, 17ß-estradiol inhibited RhoA and JNK3 activation, which were activated and peaked at 3 and/or 5 d after injury. Furthermore, administration of Rho inhibitor, PEP-1-C3 exoenzyme, inhibited RhoA and JNK3 activation, and decreased phosphorylated c-Jun level at 5 d after injury. Additionally, the attenuation of RhoA and JNK3 activation as well as oligodendrocyte cell death by 17ß-estradiol was reversed by ER antagonist, ICI182780. Our results thus indicate that 17ß-estradiol treatment improves functional recovery after SCI in part by reducing oligodendrocyte cell death via inhibition of RhoA and JNK3 activation, which were ER dependent. Furthermore, improvement of hindlimb motor function by posttreatment of 17ß-estradiol suggests its potential as a therapeutic agent for SCI patients.

Design of a Pep-1 peptide-modified liposomal nanocarrier system for intracellular drug delivery: Conformational characterization and cellular uptake evaluation.

In order to facilitate the intracellular delivery of macromolecules, Pep-1 peptide-modified liposomal (Pep1-Lipo) nanocarriers were designed and examined for their in vitro cell translocation capability. Pep-1 peptides were coupled via thiol-maleimide linkage to small unilamellar vesicles composed of phosphatidylcholine, Tween 80, and N-[4-(p-maleimidophenyl)butyryl]-phosphatidylethanolamine (MPB-PE). The amount of Pep-1 peptide conjugated to the vesicle was effectively controlled by the amounts of maleimide groups on the vesicular surface, ranging from 70 to 700 molecules per vesicle. Systems were evaluated for cell uptake capacity by monitoring entrapped fluorescence-labeled bevacizumab, a model protein for poorly permeable macromolecule, using confocal microscopy. The novel carriers rapidly bound to the cell membrane and migrated into the cells within 1 h, exhibiting better translocation of macromolecules compared to that of conventional liposomes. Cellular uptake of Pep1-Lipo was proportional to the amount of Pep-1 peptide on the liposomal surface. In conclusion, we found that the Pep1-Lipo formulation was a promising nanocarrier system for intracellular delivery of macromolecules.

Transduced human PEP-1-heat shock protein 27 efficiently protects against brain ischemic insult.

Reactive oxygen species contribute to the development of various human diseases. Ischemia is characterized by both significant oxidative stress and characteristic changes in the antioxidant defense mechanism. Heat shock protein 27 (HSP27) has a potent ability to increase cell survival in response to oxidative stress. In the present study, we have investigated the protective effects of PEP-1-HSP27 against cell death and ischemic insults. When PEP-1-HSP27 fusion protein was added to the culture medium of astrocyte and primary neuronal cells, it rapidly entered the cells and protected them against cell death induced by oxidative stress. Immunohistochemical analysis revealed that, when PEP-1-HSP27 fusion protein was intraperitoneally injected into gerbils, it prevented neuronal cell death in the CA1 region of the hippocampus in response to transient forebrain ischemia. Our results demonstrate that transduced PEP-1-HSP27 protects against cell death in vitro and in vivo, and suggest that transduction of PEP-1-HSP27 fusion protein provides a potential strategy for therapeutic delivery in various human diseases in which reactive oxygen species are implicated, including stroke.

PEN-2 overexpression induces gamma-secretase protein and its activity with amyloid beta-42 production.

PEN-2 is a component of the gamma-secretase complex, which is involved in the cleavage of the beta-amyloid precursor protein. The aim of this study was to determine the mechanism by which PEN-2 overexpression regulates gamma-secretase expression and the production of Abeta-42. In order to determine this, a hybrid gene harboring human PEN-2 was constructed, and used in the transfection of SK-N-MC human neuroepitheliomal cells. This cell line was also co-transfected with a combination of human mutant presenilin 2 (hPS2m) and APPsw. Our results indicated that (i) human PEN-2 overexpression induced an increase in gamma-secretase activity and its proteins, including PS1-CTF, APH-1, and nicastrin, thus production of Abeta-42, (ii) co-transfection of human PEN-2 with both hPS2m and APPsw exerted no more profound effects on the induction of gamma-secretase proteins and its activity than did transfection with hPEN-2 alone. Thus, PEN-2 overexpression may facilitate assembly into the more active gamma-secretase complex, and may also induce an increase in activity, thus affecting Abeta-42 production.

Differential effect of 7,12-dimethylbenz[a]anthracene on human and mouse CYP1B1 from livers of castrated transgenic mice.

Humanized transgenic mice coexpressing tetracycline-controlled transactivator (tTA) and human cytochrome P450 1B1 (CYP1B1) (hCYP1B1) have been created by this group. The aims of this study was to determine if 7,12-dimethylbenz[a]anthracene (DMBA) functions as testosterone or doxycycline in its ability to induce or reduce expression of hCYP1B1 or endogenous mouse CYP1B1 (mCYP1B1). This was tested in the livers by treating castrated transgenic males and hCYP1B1/luciferase-transfected cells with DMBA. Herein, DMBA-treated group exhibited (i) gradual reduction of hCYP1B1 expression at the transcript, protein, and activity levels but gradually induced its transcript level during DMBA release; (ii) gradual reduction of hCYP1B1 at the transcript and protein levels, as in the case of doxycycline or testosterone; (iii) gradual induction of mCYP1B1 expression at the transcript and protein levels but gradually reduced its transcript level during DMBA release. In parallel, DMBA-treated transfected cells exhibited gradual increase in luciferase activity in a time-and dose-dependent manner. Thus, castrated transgenic males or in vitro system could be useful as models for the detection of polycyclic aromatic hydrocarbons (PAHs) or environmental toxicants by measuring either hCYP1B1 or mCYP1B1 expressions.

Analysis of differentially expressed genes in early- and late-stage APPsw-transgenic and normal mice using cDNA microarray.

The complexity of Alzheimer's disease (AD) has made it difficult to examine its underlying mechanism. A gene microarray offers a solution to the complexity through a parallel analysis of most of the genes expressed in the brains from AD-transgenic mice. In our previous study, a total of 52 differentially expressed genes were identified in 18-month-old APPsw-transgenic mice compared to age-matched normal mice. We extended our work to better understand the relevant gene profiles from both early- and late-stage transgenic and normal mice. To accomplish this, cDNA microarray was used with the large-scale screening of the brain mRNA from transgenic and normal mice of 1 and 18 months of age. We identified a total of 48 genes, 6 up-regulated and 42 down-regulated, differentially expressed with a significant degree of induction and reduction in the brains from moderate 18-month-old transgenic mice compared to 1-month-old transgenic mice. In parallel, a total of 40 differentially expressed genes, 6 up-regulated and 34 down-regulated, were also found in the brains from moderate 18-month-old normal mice compared to 1-month-old normal mice. Thus, differentially expressed genes upon APPsw overexpression and the aging process are useful targets through which investigators can choose genes of particular interest. In the future, it will be necessary to study the function of differentially expressed genes, which are targets for developing drugs, using pharmacoproteomics.

Insulin-mimetic and insulin-sensitizing activities of a pentacyclic triterpenoid insulin receptor activator.

Five pentacyclic triterpenoids isolated from Campsis grandiflora were tested for insulin-mimetic and insulin-sensitizing activity. The compounds enhanced the activity of insulin on tyrosine phosphorylation of the IR (insulin receptor) beta-subunit in CHO/IR (Chinese-hamster ovary cells expressing human IR). Among the compounds tested, CG7 (ursolic acid) showed the greatest enhancement and CG11 (myrianthic acid) the least. We characterized the effect of CG7 further, and showed that it acted as an effective insulin-mimetic agent at doses above 50 mug/ml and as an insulin-sensitizer at doses as low as 1 mug/ml. Additional experiments showed that CG7 increased the number of IRs that were activated by insulin. This indicates that a major mechanism by which CG7 enhances total IR auto-phosphorylation is by promoting the tyrosine phosphorylation of additional IRs. CG7 not only potentiated insulin-mediated signalling (tyrosine phosphorylation of the IR beta-subunit, phosphorylation of Akt and glycogen synthase kinase-3beta), but also enhanced the effect of insulin on translocation of glucose transporter 4 in a classical insulin-sensitive cell line, 3T3-L1 adipocytes. The results of the present study demonstrate that a specific pentacyclic triterpenoid, CG7, exerts an insulin-sensitizing effect as an IR activator in CHO/IR cells and adipocytes. The enhancement of insulin activity by CG7 may be useful for developing a new class of specific IR activators for treatment of Type 1 and Type 2 diabetes.

Oligonucleotide-based analysis of differentially expressed genes in hippocampus of transgenic mice expressing NSE-controlled APPsw.

The complexity of Alzheimer's disease (AD) has made it difficult to examine its underlying mechanisms. A gene microarray offers a solution to the complexity through parallel analysis of most of the genes expressed in the hippocampal tissues from AD-transgenic and age-matched control littermates. This study examined the potential effect of APPsw over-expression on the modulation of genes for AD. To accomplish this, an oligonucleotide array was used with the large-scale screening of the hippocampus mRNA from 12-month-old APPsw-transgenic and control mice. There was a total of 116 differentially expressed genes, 59 up-regulated and 57 down-regulated, in the hippocampal region of the transgenic mice compared with the control mice. Initially, two of each of the down-regulated (Xlr3b and Mup3) and up-regulated genes (Serpina9 and Ccr6) were chosen for further investigation if the magnitude of change in these genes on the oligonucleotide array would correspond to those in the RT-PCR analysis from APPsw-transgenic mice. We also found that the changes in the differentially expressed genes are reliable. Thus, these genes might associate with AD neuropathology in neurodegenerative process of AD, although relevance of long lists altered genes should be evaluated in a future study.

Changes in presenilin 2-binding Wnt proteins, behavior, amyloid-beta 42, gamma-secretase activity, and testosterone sensitivity in transgenic mice coexpressing tetracycline-controlled transactivator and human mutant presenilin 2.

Nonregulatable promoters have been mainly used to produce transgenic mice that express the human genes for Alzheimer's disease (AD). The aim of this study was to produce doubly transgenic mice expressing the regulatable tet promoter-controlled transactivator (tTA) and human mutant presenilin 2 (N141I, hPS2m) genes in order to examine the AD-related phenotypes at the basal and inducible levels. To achieve this, the first lineage of the transgenic line, expressing Tet/tTA and the second lineage of transgenic mice, expressing Tet/hPS2m, were created, and the doubly transgenic mice were produced by crossing the Tet/tTA-transgenic mice with the Tet/hPS2m-transgenic mice. The doubly transgenic mice and nontransgenic littermates were then treated with or without doxycycline. The results showed that removing doxycycline from the transgenic mice resulted in the induction of the transgene, a Wnt signaling defect, behavioral impairment, elevated amyloid-beta-42 and gamma-secretase activity compared with in the group given doxycyline. Moreover, the expression levels of the hPS2m transgene decreased gradually in the transgenic males, with clear changes becoming apparent between 2 and 4 wk of age. Castrating these males resulted in an increased expression level of the hPS2m gene. This was restored to the normal levels by treatment with testosterone. Therefore, tetregulated transgenic mice can be used to examine the effect of the basal or inducible expression levels of hPS2m on the pathology of AD at the "on/off" states at any stage of development.