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Articles by Raymond Chang in JoVE

 JoVE Biology

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

1Laboratory of Neurodegenerative Diseases, Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong - HKU


JoVE 549

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Other articles by Raymond Chang on PubMed

P38 MAP Kinase is Involved in Lipopolysaccharide-induced Dopaminergic Neuronal Cell Death in Rat Mesencephalic Neuron-glia Cultures

Immune stimulants, such as the bacterial endotoxin, lipopolysaccharide (LPS), the human immunodeficiency virus-1 coat protein gp120, or beta-amyloid peptides, lead to glial activation and production of various immune mediators, such as nitric oxide (NO) and proinflammatory cytokines in the brain. These mediators appear to contribute to neuronal cell death in neurodegenerative diseases. However, the signaling pathways, which mediate the neurotoxic effect by the endotoxin, are not understood. The purpose of this study was to determine the role of mitogen-activated protein kinase (MAPK) in LPS-induced neurodegeneration using mesencephalic dopaminergic neuron/glia cultures. We have found that the p38 MAPK is important in LPS-induced death of mesencephalic neurons in rat neuron-glia mixed cultures. Upon treatment with 10 ng/ml LPS, the number of dopaminergic neurons decreased by 80% within 48 h, preceded by a significant production of NO by glia. Neuroprotection by selective inhibition of p38 MAPK activity paralleled a decrease in LPS-induced inducible nitric oxide synthase (iNOS) expression. These events were significantly reduced by the selective p38 MAPK inhibitor, SB202190, but not by the inactive analogue SB202474. Inhibition of iNOS activity and NO production by treatment with GW274150 was also neuroprotective. Although the p38 MAPK inhibitor afforded significant neuroprotection from LPS toxicity in the neuron-glia mixed culture, it failed to protect dopaminergic neurons from 6-hydroxy-dopamine-induced toxicity, which acts directly on dopaminergic neurons by inducing hydroxyl radical formation from the mitochondria. The results suggest that p38 MAPK in glia plays a significant role in the LPS-induced death of mesencephalic neurons through induction of nitric oxide synthase and resulting NO production.

Involvement of Double-stranded RNA-dependent Protein Kinase and Phosphorylation of Eukaryotic Initiation Factor-2alpha in Neuronal Degeneration

Inhibition of protein translation plays an important role in apoptosis. While double-stranded RNA-dependent protein kinase (PKR) is named as it is activated by double-stranded RNA produced by virus, its activation induces an inhibition of protein translation and apoptosis via the phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha). PKR is also a stress kinase and its levels increase during ageing. Here we show that PKR activation and eIF2alpha phosphorylation play a significant role in apoptosis of neuroblastoma cells and primary neuronal cultures induced by the beta-amyloid (Abeta) peptides, the calcium ionophore A23187 and flavonoids. The phosphorylation of eIF2alpha and the number of apoptotic cells were enhanced in over-expressed wild-type PKR neuroblastoma cells exposed to Abeta peptide, while dominant-negative PKR reduced eIF2alpha phosphorylation and apoptosis induced by Abeta peptide. Primary cultured neurons from PKR knockout mice were also less sensitive to Abeta peptide toxicity. Activation of PKR and eIF2alpha pathway by Abeta peptide are triggered by an increase in intracellular calcium because the intracellular calcium chelator BAPTA-AM significantly reduced PKR phosphorylation. Taken together, these results reveal that PKR and eIF2alpha phosphorylation could be involved in the molecular signalling events leading to neuronal apoptosis and death and could be a new target in neuroprotection.

Asian Therapies for Cancer--coming of Age

Brain Cancer: a Case of Glioblastoma Multiforme

Bioactive Polysaccharides from Traditional Chinese Medicine Herbs As Anticancer Adjuvants

To review the nature, extent, bioactivities, and clinical application of polysaccharides in Traditional Chinese Medicine (TCM) especially as adjuvants in cancer treatment.

Role of Acupuncture in the Treatment of Female Infertility

To review existing scientific rationale and clinical data in the utilization of acupuncture in the treatment of female infertility.

Phosphorylation of Eukaryotic Initiation Factor-2alpha (eIF2alpha) is Associated with Neuronal Degeneration in Alzheimer's Disease

Inhibition of protein translation is a mode of inducing neuronal apoptosis and neurodegeneration in Alzheimer's disease (AD). Phosphorylation of eukaryotic initiation factor-2alpha (eIF2alpha) terminates global protein translation and induces apoptosis. We examined whether this signaling pathway occurs in degenerating neurons of AD. Brain sections from young individuals, age-matched control individuals and AD patients were examined for immunoreactivity of phosphorylated eIF2alpha by immunohistochemical analysis. While young brain sections did not display and age-matched brain sections have mild immunoreactive positive cells, AD brain sections revealed intense immunoreactivity for phosphorylated eIF2alpha. Most of the phosphorylated eIF2alpha immunoreactive positive neurons have high immunoreactivity for phosphorylated tau using AT8 antibody. Also, intense staining of phosphorylated eIF2alpha is associated vacuoles in degenerating neurons. This phenomenon was also observed for the immunohistochemical staining of phosphorylated PKR (double-stranded RNA-dependent protein kinase), the upstream kinase for eIF2alpha. Activation of PKR-eIF2alpha pathway is considered to be pro-apoptotic. In addition, formation of autophagy is regulated by eIF2alpha kinase. Therefore, it is concluded that phosphorylation of eIF2alpha is associated with the degeneration of neurons in AD.

BAD and Bcl-2 Regulation Are Early Events Linking Neuronal Endoplasmic Reticulum Stress to Mitochondria-mediated Apoptosis

The mechanism of endoplasmic reticulum (ER)-mediated apoptosis in neurons was examined. Using primary cortical neurons, we show that nordihydroguaiaretic acid (NDGA) and brefeldin A (BFA), two ER stressors, induce early ER stress as shown by Western blotting of the eukaryotic initiation factor-2alpha (eIF2alpha), an ER stress marker. This event was associated with an enhancement of neuronal apoptosis as demonstrated by the time-dependent increase in caspase-3 activity and by nuclear fragmentation. The study of the apoptotic signaling showed the translocation of cytochrome c from the mitochondrial matrix to the cytosol. Further evaluation of the apoptotic process revealed that NDGA and BFA induced a rapid dephosphorylation of BAD and decrease expression of Bcl-2. Altogether, our results indicate that neuronal ER stress is associated with an apoptotic cascade involving the mitochondria.

Trial Spacing is a Determinant of Cue Interaction

Four conditioned lick suppression experiments with rats examined the effect of trial spacing on cue interaction. Experiments 1 and 2 found overshadowing to be eliminated with massed compound stimulus-outcome pairings and the usual trial spacing effect to be reversed with compound acquisition trials. Experiment 3 found that whether acquisition compound-outcome pairings were massed or spaced determined the effect of posttraining extinction treatment. Extinction of the overshadowing cue reduced responding following massed training and increased responding following spaced training. Extinction of the context decreased responding following massed training. Experiment 4 found the conditioning and devaluation results to be associative and stimulus specific. These results are in accord with the extended comparator hypothesis (J. C. Denniston, H. I. Savastano, & R. R. Miller, 2001).

Images in Clinical Medicine. "Corkscrew" Esophagus

Benzodiazepines As Potent and Selective Bradykinin B1 Antagonists

Antagonism of the bradykinin B(1) receptor was demonstrated to be a potential treatment for chronic pain and inflammation. Novel benzodiazepines were designed that display subnanomolar affinity for the bradykinin B(1) receptor (K(i) = 0.59 nM) and high selectivity against the bradykinin B(2) receptor (K(i) > 10 microM). In vivo efficacy, comparable to morphine, was demonstrated for lead compounds in a rodent hyperalgesia model.

Discovery of a Potent, Non-peptide Bradykinin B1 Receptor Antagonist

Bradykinin (BK) plays an important role in the pathophysiological processes accompanying pain and inflammation. Selective bradykinin B1 receptor antagonists have been shown to be anti-nociceptive in animal models and could be novel therapeutic agents for the treatment of pain and inflammation. We have explored chemical modifications in a series of dihydroquinoxalinone sulfonamides to evaluate the effects of various structural changes on biological activity. The optimization of a screening lead compound, facilitated by a homology model of the BK B1 receptor, culminated in the discovery of a potent human BK B1 receptor antagonist. Results from site-directed mutagenesis studies and experiments in an animal pain model are presented.

Backward Conditioning: Mediation by the Context

The information acquired in backward conditioning (i.e., outcome-->cue) was assessed in 3 Pavlovian lick-suppression experiments with water-deprived rats as subjects. Experiment 1 confirmed previous research that few outcome-->cue pairings made the cue into a conditioned excitor and additionally showed that massive posttraining extinction of the training context attenuated a backward-trained cue's excitatory value. Experiment 2 found that many outcome-->cue pairings made the cue into a conditioned inhibitor and that the same context manipulation attenuated this inhibitory value. Experiment 3 confirmed the observations of Experiments 1 and 2 and demonstrated that these effects of context extinction were specific to backward-trained cues conditioned in the extinguished context. These results are interpreted in terms of cue-->context and context-->outcome associations.

A Family of Highly Selective Allosteric Modulators of the Metabotropic Glutamate Receptor Subtype 5

We have identified a family of highly selective allosteric modulators of the group I metabotropic glutamate receptor subtype 5 (mGluR5). This family of closely related analogs exerts a spectrum of effects, ranging from positive to negative allosteric modulation, and includes compounds that do not themselves modulate mGluR5 agonist activity but rather prevent other family members from exerting their modulatory effects. 3,3'-Difluorobenzaldazine (DFB) has no agonist activity, but it acts as a selective positive allosteric modulator of human and rat mGluR5. DFB potentiates threshold responses to glutamate, quisqualate, and 3,5-dihydroxyphenylglycine in fluorometric Ca2+ assays 3- to 6-fold, with EC50 values in the 2 to 5 microM range, and at 10 to 100 microM, it shifts mGluR5 agonist concentration-response curves approximately 2-fold to the left. The analog 3,3'-dimethoxybenzaldazine (DMeOB) acts as a negative modulator of mGluR5 agonist activity, with an IC50 of 3 microM in fluorometric Ca2+ assays, whereas the analog 3,3'-dichlorobenzaldazine (DCB) does not exert any apparent modulatory effect on mGluR5 activity. However, DCB seems to act as an allosteric ligand with neutral cooperativity, preventing the positive allosteric modulation of mGluRs by DFB as well as the negative modulatory effect of DMeOB. None of these analogs affects binding of [3H]quisqualate to the orthosteric (glutamate) site, but they do inhibit [3H]3-methoxy-5-(2-pyridinylethynyl)pyridine binding to the site for 2-methyl-6-(phenylethynyl)-pyridine, a previously identified negative allosteric modulator. With the use of these compounds, we provide evidence that allosteric sites on GPCRs can respond to closely related ligands with a range of pharmacological activities from positive to negative modulation as well as to neutral competition of this modulation.

Upstream Signaling Pathways Leading to the Activation of Double-stranded RNA-dependent Serine/threonine Protein Kinase in Beta-amyloid Peptide Neurotoxicity

One of the hallmarks of Alzheimer's disease is extracellular accumulation of senile plaques composed primarily of aggregated beta-amyloid (Abeta) peptide. Treatment of cultured neurons with Abeta peptide induces neuronal death in which apoptosis is suggested to be one of the mechanisms. We have demonstrated previously that Abeta peptide induces activation of double-stranded RNA-dependent serine/threonine protein kinase (PKR) and phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) in neurons in vitro. Degenerating neurons in brain tissues from Alzheimer's disease patients also displayed high immunoreactivity for phosphorylated PKR and eIF2alpha. Our previous data have also indicated that PKR plays a significant role in mediating Abeta peptide-induced neuronal death, because neurons from PKR knockout mice and neuroblastoma SH-SY5Y cells stably transfected with dominant negative mutant of PKR are less susceptible to Abeta peptide toxicity. Therefore, it is important to understand how PKR is activated by Abeta peptide. We report here that inhibition of caspase-3 activity reduces phosphorylation of PKR and to a certain extent, cleavage of PKR and eIF2alpha in neurons exposed to Abeta peptide. Calcium release from the endoplasmic reticulum and activation of caspase-8 are the upstream signals modulating the caspase-3-mediated activation of PKR by Abeta peptide. Although in other systems HSP90 serves as a repressor for PKR, it is unlikely the candidate for caspase-3 to affect PKR activation in neurons after Abeta peptide exposure. Elucidation of the upstream pathways for PKR activation can help us to understand how this kinase participates in Abeta peptide neurotoxicity and to develop effective neuroprotective strategy.

Difference in MGluR5 Interaction Between Positive Allosteric Modulators from Two Structural Classes

Reduction of Calcium Release from the Endoplasmic Reticulum Could Only Provide Partial Neuroprotection Against Beta-amyloid Peptide Toxicity

Beta-amyloid (Abeta) peptide has been suggested to play important roles in the pathogenesis of Alzheimer's disease (AD). Abeta peptide neurotoxicity was shown to induce disturbance of cellular calcium homeostasis. However, whether modulation of calcium release from the endoplasmic reticulum (ER) can protect neurons from Abeta toxicity is not clearly defined. In the present study, Abeta peptide-triggered ER calcium release in primary cortical neurons in culture is modulated by Xestospongin C, 2-aminoethoxydiphenyl borate or FK506. Our results showed that reduction of ER calcium release can partially attenuate Abeta peptide neurotoxicity evaluated by LDH release, caspase-3 activity and quantification of apoptotic cells. While stress signals associated with perturbations of ER functions such as up-regulation of GRP78 was significantly attenuated, other signaling machinery such as activation of caspase-7 transmitting death signals from ER to other organelles could not be altered. We further provide evidence that molecular signaling in mitochondria play also a significant role in determining neuronal apoptosis because Abeta peptide-triggered activation of caspase-9 was not significantly reduced by attenuating ER calcium release. Our results suggest that neuroprotective strategies aiming at reducing Abeta toxicity should include molecular targets linked to ER perturbations associated with ER calcium release as well as mitochondrial stress.

Massive Preexposure and Preexposure in Multiple Contexts Attenuate the Context Specificity of Latent Inhibition

Latent inhibition, which refers to attenuated responding to a conditioned stimulus (CS) after CS-unconditioned stimulus (CS-US) pairings as a result of CS-alone presentations prior to the pairings, is often attenuated if preexposure and conditioning occur in different contexts (i.e., it is context specific). Here we report two conditioned lick suppression experiments, using rat subjects, that examined whether manipulations known to attenuate the context specificity of extinction could also eliminate the context specificity of latent inhibition. Context specificity of latent inhibition was eliminated when the CS was preexposed in multiple contexts (Experiment 1) and when the CS was massively pre-exposed in the training context alone (Experiment 2). These results and their practical implications are discussed in the framework of contemporary theories of latent inhibition.

Comparing Excitatory Backward and Forward Conditioning

Three Pavlovian lick suppression studies with rats were conducted to compare the role of the conditioning context in excitatory backward and forward conditioning. The experiments explored the possibility that excitatory backward conditioning, but not forward conditioning, is mediated by the context. That is, in excitatory backward conditioning, the conditioning context may function as an excitatory mediator, which supports second-order conditioning of the target cue. This possibility contrasts with traditional accounts, which suggests that common processes underlie excitatory backward and forward conditioning. Experiment 1 found that conditioned responding following backward conditioning was attenuated as a result of posttraining extinction of the training context, but the same manipulation elevated responding after forward conditioning. Experiments 2 and 3 found that posttraining and pretraining associative inflation of the context (presenting unsignalled USs) increased conditioned responding to the target of a backward conditioning procedure but either had no effect or reduced responding to the target of a forward conditioning procedure. Thus, excitatory backward and forward conditioning appear to differ in their dependence on the status of the conditioning context.

Generation and Characterization of a Human Bradykinin Receptor B1 Transgenic Rat As a Pharmacodynamic Model

Antagonists of the B1 bradykinin receptor (B1R) offer the promise of novel therapeutic agents for the treatment of inflammatory and neuropathic pain. However, the in vivo characterization of the pharmacodynamics of B1R antagonists is hindered by the low level of B1R expression in healthy tissue and the profound species selectivity exhibited by many compounds for the human B1R. To circumvent these issues, we generated a transgenic rat expressing the human B1R under the control of the neuron-specific enolase promoter. Membranes prepared from whole brain homogenates of heterozygous transgenic rats indicate a B1R expression level of 30 to 40 fmol/mg; there is no detectable B1R expression in control nontransgenic rats. The pharmacological profile of the B1R expressed in the transgenic rat matches that expected of the human, but not the rat receptor. The mapping of the transgene insertion site to rat chromosome 1 permitted the development of a reliable assay for the identification of homozygous transgenic rats. Significantly, homozygous transgenic rats express 2-fold more B1R than heterozygous animals. Autoradiographic analyses of tissue sections from transgenic rats reveal that the B1R is broadly expressed in both the brain and spinal cord. The human B1R expressed in the transgenic rat functions in an in vitro contractile assay and thus has the potential to elicit a functional response in vivo. Using the humanized B1R transgenic rat, an assay was developed that is suitable for the routine evaluation of a test compound's ability to occupy the human B1R in the central nervous system.

Modulation of Calcium/calmodulin Kinase-II Provides Partial Neuroprotection Against Beta-amyloid Peptide Toxicity

Beta-amyloid (Abeta) peptide-induced neurotoxicity has been implicated in the pathogenesis of Alzheimer's disease (AD). The exact mechanism by which Abeta peptides trigger neuronal death is not well defined and may be related to an abrupt increase in intracellular calcium, leading to the activation of many pro-apoptotic pathways. While modulation of intracellular calcium increase receives much attention for pharmaceutical intervention, Ca2+-mediated pro-apoptotic signalling pathways have not been systematically studied. We have reported our study on the roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in Abeta peptide neurotoxicity. By treating the primary cortical neurons exposed to Abeta peptides (Abeta(25-35) and Abeta(1-42)) with two selective CaMKII inhibitors, autocamtide-related inhibitory peptide (AIP) and KN93, Abeta peptide neurotoxicity was significantly reduced. Release of LDH and DNA fragmentation/condensation (by DAPI staining) in neurons exposed to Abeta peptides were significantly decreased in the presence of AIP and KN93. While these inhibitors significantly attenuated Abeta peptide-triggered activation of caspase-2 and caspase-3, and AIP significantly decreased the degree of tau phosphorylation of the Abeta peptide-treated neurons at early time, they could elicit partial neuroprotection only. Pharmacological inhibitor targeting calmodulin, W7, did not provide neuroprotection. Morphine, which activates CaMKII via micro receptors, augments Abeta-induced LDH release, caspase-2 and caspase-3 activities and neuronal apoptosis. Taken together, although CaMKII plays a role in Abeta peptide neurotoxicity, pharmacological inhibition cannot afford complete neuroprotection.

Reconstruction of Complex Oncologic Chest Wall Defects: a 10-year Experience

The repair of complex chest wall defects presents a challenging problem for the reconstructive surgeon. Although the majority of such defects could be repaired with the use of local and regional musculocutaneous flaps, more complicated cases require increasingly sophisticated reconstructive techniques. This study reviews the experience at a single cancer center with chest wall reconstruction over a decade. A retrospective review was undertaken for each patient who underwent chest wall reconstruction from 1992 to 2002. Patient demographics and variables, including pathologic diagnosis, extent of resection, size of defect, method of reconstruction, and outcome were evaluated. There was a total of 113 patients, 88 females and 25 males. The average age was 58 years (range, 19-88 years). The most common diagnoses were breast cancer and sarcoma. The average area of the chest wall defect after resection was 266 cm. One hundred fifty-seven musculocutaneous or muscle flaps were performed for reconstruction of the chest wall. Eleven percent of patients underwent reconstruction with autologous free tissue transfer. One hundred six patients underwent a single operation. Seven patients required a second operation for salvage of a complication. In 19 cases (15%), more than 1 flap was used simultaneously to complete the reconstruction. Eighty-four percent of the patients achieved stable chest wall reconstruction with no complications. Seven patients (4%) had partial (>10%) flap loss. The most common remaining postoperative complications were delayed wound healing (3% of patients), infection (2.5%), and hematoma (2.5%). Immediate chest wall reconstruction is safe, reliable, and can most often be accomplished with 1 operation. A variety of flaps, both single and in combination, could be used to achieve definitive coverage of the chest wall after extirpative surgery. The reconstructive choice is dependent on factors such as size of the defect, location on the chest wall, arc of rotation of the flap, and availability of recipient vessels. Based on this single institutional experience over a decade, an algorithm to chest wall reconstruction is provided.

Pharmacological Characterization and Radioligand Binding Properties of a High-affinity, Nonpeptide, Bradykinin B1 Receptor Antagonist

Compound A (N-[2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]ethyl]-2-[(2R)-1-(2-napthylsulfonyl)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetamide) is a member of a new class of aryl sulfonamide dihydroquinoxalinone bradykinin B1 receptor antagonists that should be useful pharmacological tools. Here we report on some of the pharmacological properties of compound A as well as the characterization of [35S]compound A as the first nonpeptide bradykinin B1 receptor radioligand. Compound A inhibited tritiated peptide ligand binding to the cloned human, rabbit, dog, and rat bradykinin B1 receptors expressed in CHO cells with Ki values of 0.016, 0.050, 0.56, and 29 nM, respectively. It was inactive at 10 microM in binding assays with the cloned human bradykinin B2 receptor. In functional antagonist assays with the cloned bradykinin B1 receptors, compound A inhibited agonist-induced signaling with activities consistent with the competition binding results, but had no antagonist activity at the bradykinin B2 receptor. Compound A was also found to be a potent antagonist in a rabbit aorta tissue bath preparation and to effectively block des-Arg9 bradykinin depressor responses in lipopolysaccharide-treated rabbit following intravenous administration. The binding of [35S]compound A was evaluated with the cloned bradykinin B1 receptors. In assays with human, rabbit, and dog receptors, [35S]compound A labeled a single site with Kd values of 0.012, 0.064, and 0.37 nM, respectively, and with binding site densities equivalent to those obtained using the conventional tritiated peptide ligands. Binding assays with the cloned rat bradykinin B1 receptor were not successful, presumably due to the low affinity of the ligand for this species receptor. There was no specific binding of the ligand detected in CHO cells expressing the human bradykinin B2 receptor. In assays with the cloned human bradykinin B1 receptor, the pharmacologies of the binding of [35S]compound A and [3H][Leu9]des-Arg10-kallidin were the same. The high signal-to-noise ratio obtained with [35S]compound A will allow this ligand to be a very useful tool for future investigations of the bradykinin B1 receptor.

In Vivo Evaluation of [11C]N-(2-chloro-5-thiomethylphenyl)-N'-(3-methoxy-phenyl)-N'-methylguanidine ([11C]GMOM) As a Potential PET Radiotracer for the PCP/NMDA Receptor

The development of imaging methods to measure changes in NMDA ion channel activation would provide a powerful means to probe the mechanisms of drugs and device based treatments (e.g., ECT) thought to alter glutamate neurotransmission. To provide a potential NMDA/PCP receptor PET tracer, we synthesized the radioligand [11C]GMOM (ki = 5.2 +/-0.3 nM; log P = 2.34) and evaluated this ligand in vivo in awake male rats and isoflurane anesthetized baboons. In rats, the regional brain uptake of [11C]GMOM ranged from 0.75+/-0.13% ID/g in the medulla and pons to 1.15+/-0.17% ID/g in the occipital cortex. MK801 (1 mg/kg i.v.) significantly reduced (24-28%) [11C]GMOM uptake in all regions. D-serine (10 mg/kg i.v.) increased [11C]GMOM %ID/g values in all regions (10-24%) reaching significance in the frontal cortex and cerebellum only. The NR2B ligand RO 25-6981 (10 mg/kg i.v.) reduced [11C]GMOM uptake significantly (24-38%) in all regions except for the cerebellum and striatum. Blood activity was 0.11+/-0.03 %ID/g in the controls group and did not vary significantly across groups. PET imaging in isoflurane-anesthetized baboons with high specific activity [11C]GMOM provided fairly uniform regional brain distribution volume (VT) values (12.8-17.1 ml g(-1)). MK801 (0.5 mg/kg, i.v., n = 1, and 1.0 mg/kg, i.v., n = 1) did not significantly alter regional VT values, indicating a lack of saturable binding. However, the potential confounding effects associated with ketamine induction of anesthesia along with isoflurane maintenance must be considered because both agents are known to reduce NMDA ion channel activation. Future and carefully designed studies, presumably utilizing an optimized NMDA/PCP site tracer, will be carried out to further explore these hypotheses. We conclude that, even though [11C]GMOM is not an optimized PCP site radiotracer, its binding is altered in vivo in awake rats as expected by modulation of NMDA ion channel activity by MK801, D-serine or RO 25-6981. The development of higher affinity NMDA/PCP site radioligands is in progress.

Characterization of Polysaccharides from the Flowers of Nerium Indicum and Their Neuroprotective Effects

Degeneration of neurons is a key problem in Alzheimer's disease (AD) and neuroprotection is a possible way to safeguard neurons from neurodegeneration. Polysaccharides isolated from Chinese medicinal herbs have been investigated extensively for their anti-tumor and immune stimulating effects. Yet, little is known about the effects of polysaccharides in neurons. Recently, two pure polysaccharides isolated from the flowers of Nerium indicum were shown to stimulate proliferation and differentiation of PC12 pheochromocytoma cells, an effect similar to that observed from nerve growth factor. In this notion, it is hypothesized that polysaccharides isolated from the flowers of N. indicum could exhibit beneficial effects in neurons. In this study, we isolated, characterized and investigated two new polysaccharides from the flowers of N. indicum for their neuroprotective effects on neurons against serum-deprivation and beta-amyloid (Abeta) peptide toxicity in primary rat cortical neuronal cultures. Pretreatment of the polysaccharides significantly reduced the number of apoptotic neurons revealed by DAPI staining when neurons were exposed to serum-free medium. Besides, the polysaccharides could also decrease the activity of caspase-3 triggered by Abeta peptides. Western blot analysis indicated that polysaccharides stimulated the phosphorylation of PDK-1 (Serine 241) and Akt (Threonine 308). In conclusion, the polysaccharides J2, J3 and J4 isolated from N. indicum provide a lead for future development of neuroprotective agent against neuronal death in neurodegenerative diseases and the neuroprotective mechanism may primarily rely on activation of Akt survival signaling pathway.

Isolated Cortical Venous Thrombosis Presenting As Subarachnoid Hemorrhage: a Report of Three Cases

Cortical venous thrombosis (CVT) without concomitant dural sinus thrombosis is an uncommon disorder. Isolated CVT usually manifests on imaging studies as focal parenchymal hemorrhage or edema. We report three cases of isolated CVT that presented with unilateral, localized subarachnoid hemorrhage without parenchymal involvement.

2,3-diaminopyridine Bradykinin B1 Receptor Antagonists

Bradykinin B1 receptor antagonists embody a potentially novel approach for the treatment of chronic pain and inflammation. A series of 2,3-diaminopyridine B1 antagonists was optimized to have sub-nanomolar affinity and good pharmacokinetic properties. Lead compounds were shown to exhibit good efficacy in rabbit in vivo models of pain and inflammation.

Immune Modulatory Effects of Prunella Vulgaris L

Prunella vulgaris L. (Labiatae) is a perennial plant known as 'self-heal' in Western herbal medicine. It has a wide array of biological effects exhibiting numerous therapeutic potentials. Its anti-microbial effects including anti-viral and anti-bacterial effects are, presently, receiving increasing attention. While its anti-viral effects are attributed mainly to the inhibition of virus replication, the biological mechanisms of its anti-bacterial effects or actions remain unknown. In view of the fact that polysaccharides isolated from medicinal herbs often function as biological response modifier of body immunity, we hypothesized that the anti-microbial effect of polysaccharides isolated from P. vulgaris is probably also mediated via immune modulation. We have isolated four polysaccharides containing fractions from P. vulgaris, one of the fractions, PV2, could markedly stimulate the production of superoxide and nitrite representing nitric oxide from murine macrophage RAW264.7 and brain macrophage BV2 cells. The amount of nitrite and superoxide produced after PV2 stimulation was as high as that seen in stimulation using bacterial endotoxin lipopolysaccharide (LPS), and this stimulatory response is dose-dependent. In addition to monocyte/macrophage, PV2 also stimulated the proliferation of splenocytes. In this study, we have shown that the polysaccharides isolated from P. vulgaris have marked immune stimulatory effects, which may bring about the anti-microbial effects of P. vulgaris.

2,3-Diaminopyridine As a Platform for Designing Structurally Unique Nonpeptide Bradykinin B1 Receptor Antagonists

A novel class of 2,3-diaminopyridine bradykinin B1 receptor antagonists is disclosed. Structure-activity relationship studies (SARs) that led to compounds with significantly improved potency and pharmacokinetic properties relative to the lead compound are described.

Stable Expression of EBERs in Immortalized Nasopharyngeal Epithelial Cells Confers Resistance to Apoptotic Stress

Epstein-Barr virus (EBV) infection is closely associated with the development of nasopharyngeal carcinoma (NPC). The EBV-encoded RNAs (EBERs) are the most abundant EBV transcripts (about 10(7) copies per cell) in EBV infected cells. However, the cellular function of EBER expression, particularly in nasopharyngeal epithelial cells, remains poorly understood. EBERs acquire secondary structures analogous to double-stranded RNA (dsRNA) and may bind to the double-stranded RNA-dependent protein kinase (PKR) and interfere with its function. Activation of PKR involves autophosphorylation resulting in protein synthesis inhibition and cellular apoptosis. Induction of cellular apoptosis by activation of PKR may be an antiviral response adopted by virally infected cells. We have examined the functional properties of EBER expression in an immortalized nasopharyngeal epithelial cell line (NP69). Expression of EBERs was achieved by transfecting the NP69 cells with an EBER-expressing plasmid, pESK10. The EBER-expressing NP69 cells attained a higher growth rate compared to cells transfected with control plasmid (pcDNA3). However, the EBER-expressing NP69 cells did not form colonies in soft agar and were non-tumorigenic in nude mice. To investigate if EBERs may protect the nasopharyngeal epithelial cells from apoptotic insults, we treated the EBER-expressing NP69 cells with a dsRNA analogue, poly(I).poly(C) (pIC), to activate PKR in cells and examined for their responses. Lower level of PKR phosphorylation and elevation of Bcl-2 were observed in EBER-expressing NP69 cells. In addition, other apoptotic markers including the cleaved forms of caspase-3 and poly(ADP)ribose polymerase (PARP) were found to be lower in EBER-expressing NP69 cells after treatment with pIC. Lower phosphorylation levels of p38 MAPK (mitogen-activated protein kinase) and c-jun were also observed in EBER-expressing NP cells. Our results suggest that EBER expression may confer an apoptotic-resistant phenotype in immortalized nasopharyngeal epithelial cells.

Neuroprotective Effects of Anti-aging Oriental Medicine Lycium Barbarum Against Beta-amyloid Peptide Neurotoxicity

As aged population dramatically increases in these decades, efforts should be made on the intervention for curing age-associated neurodegenerative diseases such as Alzheimer's disease (AD). Natural plant extracts of Lycium barbarum are well-known to exhibit anti-aging effects. We therefore hypothesized that they exhibit neuroprotective effects against toxins in aging-related neurodegenerative diseases. In this study, we aimed to investigate whether extracts from L. barbarum have neuroprotective effects against toxicity of fibrillar Abeta(1-42) and Abeta(25-35) fragments. Primary rat cortical neurons exposed to Abeta peptides resulted in apoptosis and necrosis. Pre-treatment with extract isolated from L. barbarum significantly reduced the release of lactate dehydrogenase (LDH). In addition, it attenuated Abeta peptide-activated caspases-3-like activity. The extract elicited a typical dose-dependent neuroprotective effect. Effective dosage of this extract was wider than that of a well-known western neuroprotective medicine lithium chloride (LiCl). We have further examined the underlying mechanisms of the neuroprotective effects. In agreement with other laboratories, Abeta peptides induce a rapid activation of c-Jun N-terminal kinase (JNK) by phosphorylation. Pre-treatment of aqueous extract markedly reduced the phosphorylation of JNK-1 (Thr183/Tyr185) and its substrates c-Jun-I (Ser 73) and c-Jun-II (Ser 63). Taken together, we have proved our hypothesis by showing neuroprotective effects of the extract from L. barbarum. Study on anti-aging herbal medicine like L. barbarum may open a new therapeutic window for the prevention of AD.

Immune Modulatory Effects of Prunella Vulgaris L. on Monocytes/macrophages

Prunella vulgaris L. (Labiatae), a popular Western and Chinese herbal medicine, has long been associated with anti-viral and anti-bacterial effects. While its anti-viral effects are attributed mainly to the inhibition of virus replication, the biological mechanisms of its anti-bacterial effects remain unknown. As a biological response modifier (BRM), the polysaccharides isolated from P. vulgaris have been shown to up-regulate the immune responses of monocytes/macrophages. However, the immune stimulatory effects seem to contradict its well-known anti-inflammatory properties. We hypothesized that the anti-microbial effects exhibited by the polysaccharides isolated from P. vulgaris encompass both anti-inflammatory and immune stimulatory effects. One of the polysaccharide fractions PV2IV markedly stimulated the production of superoxide and nitrite representing nitric oxide from murine macrophage RAW264.7 and brain macrophage BV2 cells. The amount of nitrite and superoxide produced after PV2IV stimulation was as high as that stimulated by bacterial endotoxin lipopolysaccharide (LPS) in a dose-dependent manner. In addition, PV2IV also increased cellular protein levels of inducible nitric oxide synthase (iNOS) and mRNA for tumor necrosis factor-alpha (TNFalpha). Similar to the effects of a high dose of LPS, the fraction PV2 could trigger activation-induced cell death (AICD) by stimulating caspase-3 activity and reduction of MTT uptake in monocytes/macrophages. These results may help our understanding of the molecular mechanism of P. vulgaris, which exhibited both immune stimulatory and anti-inflammatory effects against microbial invasion.

Inhibition of Acute Nociceptive Responses in Rat Spinal Cord by a Bradykinin B1 Receptor Antagonist

This study used behavioural and in vivo electrophysiological paradigms to examine the effects of systemic and spinal administration of a bradykinin B1 receptor antagonist, compound X, on acute nociceptive responses in the rat. In behavioural experiments, compound X significantly increased the latency to withdraw the hindpaw from a radiant heat source after both intravenous and intrathecal administration, without affecting motor performance on the rotarod. In electrophysiological experiments, both intravenous and direct spinal administration of compound X attenuated the responses of single dorsal horn neurones to noxious thermal stimulation of the hindpaw. These data show that the antinociceptive effects of a bradykinin B1 receptor antagonist are mediated, at least in part, at the level of the spinal cord and suggest a role for spinal bradykinin B1 receptors in acute nociception.

Novel Neuroprotective Effects of the Aqueous Extracts from Verbena Officinalis Linn

Verbena officinialis Linn. (Verbenaceae) is a perennial plant which has been used as herbal medicine or health supplement in both Western and Eastern countries for centuries. It has been used to treat acute dysentery, enteritis, amenorrhea and depression. In view of its wide array of biological effects, we hypothesized that V. officinalis can exert cytoprotective effects on cells of the central nervous system. Pre-treatment of aqueous extracts of V. officinalis significantly attenuated the toxicity of beta-amyloid (Abeta) peptide and reducing agent dithiothreitol in primary cultures of cortical neurons. As extracellular accumulation of Abeta peptide is an important cytotoxic factor involved in Alzheimer's disease (AD), we have further explored its neuroprotective effect against Abeta. Treatment of V. officinalis attenuated Abeta-triggered DEVD- and VDVAD-cleavage activities in a dose-dependent manner. Further studies elucidated that phosphorylation of both interferon-inducing protein kinase (PKR) and c-Jun N-terminal kinase (JNK) was attenuated in Abeta-treated neurons. Taken together, we have proved our hypothesis by showing the novel neuroprotective effects of V. officinalis. As V. officinalis has long been used for many years to be a folk medicine, our study may provide a lead for its potential to be a neuroprotective agent against neuronal loss in AD.

Cyclopropylamino Acid Amide As a Pharmacophoric Replacement for 2,3-diaminopyridine. Application to the Design of Novel Bradykinin B1 Receptor Antagonists

Antagonism of the bradykinin B1 receptor represents a potential treatment for chronic pain and inflammation. Novel antagonists were designed that display low-nanomolar affinity for the human bradykinin B1 receptor and good bioavailability in the rat.

Beta-amyloid Peptides Induces Neuronal Apoptosis Via a Mechanism Independent of Unfolded Protein Responses

Accumulation of beta-amyloid (Abeta) peptides in senile plaques is one of the pathological hallmarks in Alzheimer's disease (AD), which can trigger apoptosis. We have previously demonstrated that Abeta triggered calcium release from the ER. Depletion of ER Ca(2+) ions has been reported leading to unfolded protein responses (UPR). While hypothesis has been made about UPR and neurodegeneration in AD, little is known about the effects of extracellular accumulation of Abeta on UPR. We have shown previously that activation of PKR in Abeta-triggered apoptosis. Since UPR can trigger PKR, our study aims to elucidate whether extracellular accumulation of Abeta peptides induce UPR in cultured neurons. Our results showed that Abeta could not trigger UPR signalings including phosphorylation of PERK, alternative cleavage of xbp-1 mRNA and induction of transcription of xbp-1 and Gadd153. Taken together, our results suggest that extracellular accumulation of Abeta peptides induce apoptosis via a mechanism independent of UPR.

In Vivo Evaluation in Rats of [(18)F]1-(2-fluoroethyl)-4-[(4-cyanophenoxy)methyl]piperidine As a Potential Radiotracer for PET Assessment of CNS Sigma-1 Receptors

Sigma-1 receptors are expressed throughout the mammalian central nervous system (CNS) and are implicated in several psychiatric disorders, including schizophrenia and depression. We have recently evaluated the high-affinity (K(D)=0.5+/-0.2 nM, log P=2.9) sigma-1 receptor radiotracer [(18)F]1-(3-fluoropropyl)-4-(4-cyanophenoxymethyl)piperidine, [(18)F]FPS, in humans. In contrast to appropriate kinetics exhibited in baboon brain, in the human CNS, [(18)F]FPS does not reach pseudoequilibrium by 4 h, supporting the development of a lower-affinity tracer [Waterhouse RN, Nobler MS, Chang RC, Zhou Y, Morales O, Kuwabara H, et al. First evaluation of the sigma-1 receptor radioligand [(18)F]1-3-fluoropropyl-4-((4-cyanophenoxy)-methyl)piperidine ([(18)F]FPS) in healthy humans. Neuroreceptor Mapping 2004, July 15-18th, Vancouver, BC Canada 2004]. We describe herein the in vivo evaluation in rats of [(18)F]1-(2-fluoroethyl)-4-[(4-cyanophenoxy)methyl]piperidine ([(18)F]SFE) (K(D)=5 nM, log P=2.4), a structurally similar, lower-affinity sigma-1 receptor radioligand.

Cytoprotective Effects of Lycium Barbarum Against Reducing Stress on Endoplasmic Reticulum

Chinese medicinal herbs have been consumed for thousands of years for the purpose of healthy aging. Lycium barbarum is valued in Chinese culture for its benefits to anti-aging, vision, kidney and liver. Recent studies showed that extracts from L. barbarum possess biological activities including anti-aging, anti-tumor, immune-stimulatory and cytoprotection. Most of these studies emphasized that the protective function of L. barbarum is due to its anti-oxidative effects. We have previously demonstrated that extract from L. barbarum can protect neurons against beta-amyloid (Abeta) peptide-induced apoptosis. Since Abeta toxicity may be mediated via oxidative stress, it is still unclear whether the extract from L. barbarum is a simple anti-oxidant exhibiting cytoprotective effects. We hypothesized that extract from L. barbarum is not simply an anti-oxidant in order to function as a neuroprotective agent. The aim of this study is to investigate whether the extract from L. barbarum (LBG) protect neurons via mechanisms independent of anti-oxidative effects. Using a reducing agent, dithiothreitol (DTT), we found that LBG exhibits cytoprotective effects against reducing stress by lowering the DTT-induced LDH release and caspase-3 activity. DTT can trigger endoplasmic reticulum (ER) stress leading to PKR-like ER kinase (PERK) activation. We also showed that LBG attenuates DTT-induced PERK phosphorylation. The extract from L. barbarum is not simply an anti-oxidant; it can also exhibit cytoprotective effects against reducing stress by DTT.

Design, Synthesis, and in Vivo Efficacy of Glycine Transporter-1 (GlyT1) Inhibitors Derived from a Series of [4-phenyl-1-(propylsulfonyl)piperidin-4-yl]methyl Benzamides

Preclinical Acute Toxicity Studies and Dosimetry Estimates of the Novel Sigma-1 Receptor Radiotracer, [18F]SFE

[(18)F]1-(2-Fluoroethyl)-4-[(4-cyanophenoxy)methyl]piperidine ([(18)F]SFE) is a novel, selective, high-affinity sigma-1 receptor radioligand that has been preclinically well characterized in rodents. To support an investigational new drug (IND) application for the first evaluation of [(18)F]SFE in humans, single-organ and whole-body radiation adsorbed doses associated with [(18)F]SFE injection were estimated from rat distribution data. In addition, single- and multiple-dose toxicity studies were conducted in rabbits and in dogs. Multiple-dose toxicity studies in rabbits and single-dose toxicity studies in beagles suggest at least a 100-fold safety margin for humans studies at a mass dose limit of 4.0 mug per intravenous injection, based on the combined no observable adverse effect levels (NOAEL, mg/m(2)) measured in these species. Radiation dosimetry estimates obtained from rat biodistribution analyses of [(18)F]SFE suggest that most tissues would receive about 0.010-0.020 mGy/MBq, while the adrenal glands, brain, bone, liver, lungs, and spleen would receive slightly higher doses (0.024-0.044 mGy/MBq). The adrenal glands were identified as the critical organ, because they received the highest adsorbed radiation dose. The total exposure resulting from a 5 mCi administration of [(18)F]SFE is well below the FDA-defined limits for yearly cumulative and per-study exposures to research participants. These combined results support the expectation that [(18)F]SFE will be safe for use in human positron emission tomography (PET) imaging studies with the administration of 5 mCi and a mass dose equal to or less than 4.0 mug SFE per injection.

Significance of Molecular Signaling for Protein Translation Control in Neurodegenerative Diseases

It has long been known that protein synthesis is inhibited in neurological disorders. Protein synthesis includes protein transcription and translation. While many studies about protein transcription have been done in the last decade, we are just starting to understand more about the impact of protein translation. Protein translation control can be accomplished at the initiation or elongation steps. In this review, we will focus on translation control at initiation. Neurons have long neurites in which proteins have to be transported from the cell body to the end of the neurite. Since supply of proteins cannot meet the need of neuronal activity at the spine, protein locally translated at the spine will be a good solution to replace the turnover of proteins. Therefore, local protein translation is an important mechanism to maintain normal neuronal functions. In this notion, we have to separate the concept of global and local protein translation control. Both global and local protein translation control modulate normal neuronal functions from development to cognitive functions. Increasing lines of evidence show that they also play significant roles in neurodegenerative diseases, e.g. neuronal apoptosis, synaptic degeneration and autophagy. We summarize all the evidence in this review and focus on the control at initiation. The new live-cell imaging technology together with photoconvertible fluorescent probes allows us to investigate newly translated proteins in situ. Protein translation control is another line to modulate neuronal function in neuron-neuron communication as well as in response to stress in neurodegenerative diseases.

Neuroprotective Effects of Lycium Barbarum Lynn on Protecting Retinal Ganglion Cells in an Ocular Hypertension Model of Glaucoma

Glaucoma is one of the major neurological disorders in eye leading to irreversible blindness in elderly. Increase in intraocular pressure (IOP) has been considered to be the major risk factor for the progressive loss of retinal ganglion cells (RGCs) in retina. While attenuation of IOP has been a major pharmaceutical target, reduction of IOP cannot prevent progressive loss of RGCs. In this regard, urgent need for alternative treatment has to be investigated. Anti-aging medicinal herb Lycium barbarum L. has been used for centuries in Eastern World to protect the eyes and maintain good health. Using an ocular hypertension (OH) model in rat by laser photocoagulation of episcleral and limbal veins, we attempted to investigate whether L. barbarum can promote RGCs survival against elevated IOP. Oral administration of L. barbarum in Sprague-Dawley rats (250-280 g) significantly reduced the loss of RGCs, although elevated IOP was not significantly altered. Rats fed with the 1 mg/kg extract could nearly totally escape from pressure-induced loss of RGCs. In conclusion, this is the first in vivo report showing the therapeutic function of L. barbarum against neurodegeneration in the retina of rat OH model. The results demonstrate that this extract may be a potential candidate for the development of neuroprotective drug against the loss of RGCs in glaucoma.

Potent Bradykinin B1 Receptor Antagonists: 4-substituted Phenyl Cyclohexanes

Selective bradykinin (BK) B(1) receptor antagonists have been shown to be antinociceptive in animal models and could be novel therapeutic agents for the treatment of pain and inflammation. Elucidation of the structure-activity relationships of the biphenyl moiety of the lead compound 1 provided a potent new structural class of BK B(1) receptor antagonists.

In Vitro and in Vivo Binding of Neuroactive Steroids to the Sigma-1 Receptor As Measured with the Positron Emission Tomography Radioligand [18F]FPS

Sigma-1 receptors are widely expressed in the mammalian brain and also in organs of the immune, endocrine and reproductive systems. Based on behavioral and pharmacological assessments, sigma-1 receptors are important in memory and cognitive processes, and are thought to be involved in specific psychiatric illnesses, including schizophrenia, depression, and drug addiction. It is thought that specific neuroactive steroids are endogenous ligands for these sites. In addition, several sigma-1 receptor binding steroids including progesterone, dihydroepiandrosterone (DHEA), and testosterone are being examined clinically for specific therapeutic purposes; however, their mechanisms of action have not been clearly defined. We previously described the high affinity sigma-1 receptor selective PET tracer [(18)F]FPS. This study examines the effect of neuroactive steroids on [(18)F]FPS binding in vitro and in vivo. Inhibition constants were determined in vitro for progesterone, testosterone, DHEA, estradiol, and estriol binding to the [(18)F]FPS labeled receptor. The affinity order (K(i) values) for these steroids ranged from 36 nM for progesterone to >10,000 nM for estrodiol and estriol. Biodistribution studies revealed that i.v. coadministration of progesterone (10 mg/kg), testosterone (20 mg/kg), or DHEA (20 mg/kg) significantly decreased [(18)F]FPS uptake (%ID/g) by up to 50% in nearly all of eight brain regions examined. [(18)F]FPS uptake in several peripheral organs that express sigma-1 receptors (heart, spleen, muscle, lung) was also reduced (54-85%). These studies clearly demonstrate that exogenously administered steroids can occupy sigma-1 receptors in vivo, and that [(18)F]FPS may provide an effective tool for monitoring sigma-1 receptor occupancy of specific therapeutic steroids during clinical trials.

New Polysaccharide from Nerium Indicum Protects Neurons Via Stress Kinase Signaling Pathway

Most of the polysaccharides purified from Chinese medicinal herbs showed anti-tumor and immune-stimulating effects. However, little is known about their effects on neuroprotection. Our previous study has demonstrated that polysaccharides (J2, J3 and J4) isolated from the flowers of Nerium indicum (Oleander) exert partial protection in cortical neurons stressed by beta-amyloid (Abeta) peptides or deprivation of nutrition from serum. In this study, we have isolated and characterized a new polysaccharide from the flowers of N. indicum (named as J6) and aimed to investigate its neuroprotective effects against Abeta-induced apoptosis. Pretreatment of the polysaccharide J6 significantly decreased the activity of caspase-3 as well as the cytotoxicity triggered by Abeta peptides in a dose-dependent manner. In contrast to the activation of survival signaling such as Akt found in J2, J3 and J4 fractions, neuroprotective effects of J6 markedly inhibited Abeta peptide-stimulated phosphorylation of c-Jun N-terminal kinase (JNK-1) as determined by Western blot analysis. Taken together, the polysaccharide J6 isolated from the flowers of N. indicum can serve as potential neuroprotective agent against neuronal death in Alzheimer's disease and the neuroprotective mechanism may primarily rely on inactivation of JNK signaling pathway.

Characterizing the Neuroprotective Effects of Alkaline Extract of Lycium Barbarum on Beta-amyloid Peptide Neurotoxicity

Lycium barbarum is an oriental medicinal herb that has long been used for its anti-aging and cell-protective properties. Previous studies have shown that aqueous extracts from L. barbarum exhibit neuroprotection via inhibiting pro-apoptotic signaling pathways. Other active components can also be accomplished by novel alkaline extraction method, which may give different profiles of water-soluble components. We hypothesize that another active component obtained by alkaline extraction method exerts different biological mechanisms to protect neurons. In this study, we aim to examine the neuroprotective effects from the alkaline extract of L. barbarum, namely LBB, to attenuate beta-amyloid (Abeta) peptide neurotoxicity. Primary cortical neurons were exposed to Abeta-peptides inducing apoptosis and neuronal cell death. Pretreatment of LBB significantly reduced the level of lactate dehydrogenase (LDH) release and the activity of caspase-3 triggered by Abeta. "Wash-out" procedures did not reduce its neuroprotective effects, suggesting that LBB may not bind directly to Abeta. We have further isolated three subfractions from LBB, namely LBB-0, LBB-I and LBB-II. LBB-I and LBB-II showed differential neuroprotective effects. Western blot analysis demonstrated that LBB-I and LBB-II markedly enhanced the phosphorylation of Akt. Taken together, our results suggested that the glycoconjugate isolated from novel alkaline extraction method can open up a new avenue for drug discovery in neurodegenerative diseases.

Characterization of the Effects of Anti-aging Medicine Fructus Lycii on Beta-amyloid Peptide Neurotoxicity

Alzheimer's disease (AD) is an age-related neurodegenerative disease. There are increasing lines of evidence showing that the molecular signaling pathways in aged cells are altered so that cells are susceptible to injury. We and other laboratories have demonstrated the significant involvement of double-stranded RNA-dependent protein kinase (PKR) in beta-amyloid (A beta) peptide neurotoxicity and in AD. Fructus lycii (the fruit of Lycium barbarum) has long been used in oriental medicine as an anti-aging agent. Our previous studies demonstrated that the aqueous extract isolated from L. barbarum exhibited significant protection on cultured neurons against harmful chemical toxins such as A beta and dithiothreitol. We also showed that the polysaccharide-containing extract (LBP) from L. barbarum exhibited neuroprotective effects in the retina against ocular hypertension in a laser-induced glaucoma animal model. In this study, we aimed to investigate whether LBP can elicit neuroprotection to neurons stressed by A beta peptides. Furthermore, we planned to isolate and identify the neuroprotective agent from LBP using chromatographic methods. Our results showed that pretreatment of LBP effectively protected neurons against A beta-induced apoptosis by reducing the activity of both caspase-3 and -2, but not caspase-8 and -9. A new arabinogalactan-protein (LBP-III) was isolated from LBP and attenuated A beta peptide-activated caspase-3-like activity. LBP-III markedly reduced the phosphorylation of PKR triggered by A beta peptide. Since the phosphorylation state of PKR increased with age, reduction of its phosphorylation triggered by A beta peptide may implicate that LBP-III from Fructus lycii is a potential neuroprotective agent in AD. As herbal medicine has received increasing attention for the treatment of AD, our study will open a window for the development of a neuroprotective agent for anti-aging from Chinese medicine.

Use of Anti-aging Herbal Medicine, Lycium Barbarum, Against Aging-associated Diseases. What Do We Know So Far?

Lycium barbarum (Gouqizi, Fructus Lycii, Wolfberry) is well known for nourishing the liver, and in turn, improving the eyesight. However, many people have forgotten its anti-aging properties. Valuable components of L. barbarum are not limited to its colored components containing zeaxanthin and carotene, but include the polysaccharides and small molecules such as betaine, cerebroside, beta-sitosterol, p-coumaric, and various vitamins. Despite the fact that L. barbarum has been used for centuries, its beneficial effects to our bodies have not been comprehensively studied with modern technology to unravel its therapeutic effects at the biochemical level. Recently, our laboratory has demonstrated its neuroprotective effects to counter neuronal loss in neurodegenerative diseases. Polysaccharides extracted from L. barbarum can protect neurons against beta-amyloid peptide toxicity in neuronal cell cultures, and retinal ganglion cells in an experimental model of glaucoma. We have even isolated the active component of polysaccharide which can attenuate stress kinases and pro-apoptotic signaling pathways. We have accumulated scientific evidence for its anti-aging effects that should be highlighted for modern preventive medicine. This review is to provide background information and a new direction of study for the anti-aging properties of L. barbarum. We hope that new findings for L. barbarum will pave a new avenue for the use of Chinese medicine in modern evidence-based medicine.

Alpha-hydroxy Amides As a Novel Class of Bradykinin B1 Selective Antagonists

Antagonism of the bradykinin B(1) receptor represents a potential treatment for chronic pain and inflammation. Novel antagonists incorporating alpha-hydroxy amides were designed that display low-nanomolar affinity for the human bradykinin B(1) receptor and good bioavailability in the rat and dog. In addition, these functionally active compounds show high passive permeability and low susceptibility to phosphoglycoprotein mediated efflux, predictive of good CNS exposure.

Antagonizing Beta-amyloid Peptide Neurotoxicity of the Anti-aging Fungus Ganoderma Lucidum

Ganoderma lucidum (Leyss. ex Fr.) Karst. (Lingzhi) is a medicinal fungus used clinically in many Asian countries to promote health and longevity. Synaptic degeneration is another key mode of neurodegeneration in Alzheimer's disease (AD). Recent studies have shown the loss of synaptic density proteins in each individual neuron during the progression of AD. It was recently reported that beta-amyloid (Abeta) could cause synaptic dysfunction and contribute to AD pathology. In this study, we reported that aqueous extract of G. lucidum significantly attenuated Abeta-induced synaptotoxicity by preserving the synaptic density protein, synaptophysin. In addition, G. lucidum aqueous extract antagonized Abeta-triggered DEVD cleavage activities in a dose-dependent manner. Further studies elucidated that phosphorylation of c-Jun N-terminal kinase, c-Jun, and p38 MAP kinase was attenuated by G. lucidum in Abeta-stressed neurons. Taken together, the results prove a hypothesis that anti-aging G. lucidum can prevent harmful effects of the exterminating toxin Abeta in AD.

2-Aminobenzophenones As a Novel Class of Bradykinin B1 Receptor Antagonists

Selective bradykinin (BK) B 1 receptor antagonists could be novel therapeutic agents for the treatment of pain and inflammation. Elucidation of the structure activity relationships of the structurally novel HTS lead compound 1 provided potent hBK B 1 receptor antagonists with excellent receptor occupancy in the CNS of hBK B 1 transgenic rats.

Dietary Oxyresveratrol Prevents Parkinsonian Mimetic 6-hydroxydopamine Neurotoxicity

Oxyresveratrol (OXY) is a polyhydroxylated stilbene existing in mulberry. Increasing lines of evidence have shown its neuroprotective effects against Alzheimer disease and stroke. However, little is known about its neuroprotective effect in Parkinson disease (PD). Owing to its antioxidant activity, blood-brain barrier permeativity, and water solubility, we hypothesized that OXY may exert neuroprotective effects against parkinsonian mimetic 6-hydroxydopamine (6-OHDA) neurotoxicity. Neuroblastoma SH-SY5Y cells have long been used as dopaminergic neurons in PD research. We found that both pretreatment and posttreatment with OXY on SH-SY5Y cells significantly reduced the release of lactate dehydrogenase, the activity of caspase-3, and the generation of intracellular reactive oxygen species triggered by 6-OHDA. Compared to resveratrol, OXY exhibited a wider effective dosage range. We proved that OXY could penetrate the cell membrane by HPLC analysis of cell extracts. These results suggest that OXY may act as an intracellular antioxidant to reduce oxidative stress induced by 6-OHDA. Western blot analysis demonstrated that OXY markedly attenuated 6-OHDA-induced phosphorylation of JNK and c-Jun. Furthermore, we proved that OXY increased the basal levels of SIRT1, which may disclose new pathways accounting for the neuroprotective effects of OXY. Taken together, our results suggest OXY, a dietary phenolic compound, as a potential nutritional candidate for protection against neurodegeneration in PD.

Dexras1 Interacts with FE65 to Regulate FE65-amyloid Precursor Protein-dependent Transcription

FE65 is an adaptor protein that binds to and forms a transcriptionally active complex with the gamma-secretase-derived amyloid precursor protein (APP) intracellular domain. The regulatory mechanisms of FE65-APP-mediated transcription are still not clear. In this report, we demonstrate that Dexras1, a Ras family small G protein, binds to FE65 PTB2 domain and potently suppresses the FE65-APP-mediated transcription. The suppression is not via competition for binding of FE65 between Dexras1 and APP because the two proteins can simultaneously bind to the FE65 PTB2 domain. Phosphorylation of FE65 tyrosine 547 within the PTB2 domain has been shown to enhance FE65-APP-mediated transcription but not to influence binding to APP. Here we find that this phosphorylation event reduces the binding between Dexras1 and FE65. We also demonstrate that Dexras1 inhibits the FE65-APP-mediated transcription of glycogen synthase kinase 3beta (GSK3 beta). Moreover, small interfering RNA knockdown of Dexras1 enhances GSK3 beta expression and increases phosphorylation of Tau, a GSK3 beta substrate. Thus, Dexras1 functions as a suppressor of FE65-APP-mediated transcription, and FE65 tyrosine 547 phosphorylation enhances FE65-APP-mediated transcription, at least in part, by modulating the interaction between FE65 and Dexras1. These findings reveal a novel regulatory mechanism for FE65-APP-mediated signaling.

Effects of All-trans-retinoic Acid on Human SH-SY5Y Neuroblastoma As in Vitro Model in Neurotoxicity Research

Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line which has been used as an in vitro model for neurotoxicity experiments. Although the neuroblastoma is usually differentiated by all-trans-retinoic acid (RA), both RA-differentiated and undifferentiated SH-SY5Y cells have been used in neuroscience research. However, the changes in neuronal properties triggered by RA as well as the subsequent responsiveness to neurotoxins have not been comprehensively studied. Therefore, we aim to re-evaluate the differentiation property of RA on this cell line. We hypothesize that modulation of signaling pathways and neuronal properties during RA-mediated differentiation in SH-SY5Y cells can affect their susceptibility to neurotoxins. The differentiation property of RA was confirmed by showing an extensive outgrowth of neurites, increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin and synaptic associated protein-97, and decreased expression of inhibitor of differentiation-1. While undifferentiated SH-SY5Y cells were susceptible to 6-OHDA and MPP+, RA-differentiation conferred SH-SY5Y cells higher tolerance, potentially by up-regulating survival signaling, including Akt pathway as inhibition of Akt removed RA-induced neuroprotection against 6-OHDA. As a result, the real toxicity cannot be revealed in RA-differentiated cells. Therefore, undifferentiated SH-SY5Y is more appropriate for studying neurotoxicity or neuroprotection in experimental Parkinson's disease research.

Low Molecular Weight Abeta Induces Collapse of Endoplasmic Reticulum

The endoplasmic reticulum (ER) is a dynamic multifunction organelle that is responsible for Ca(2+) homeostasis, protein folding, post-translational modification, protein degradation, and transportation of nascent proteins. Disruption of ER architecture might affect the normal physiology of the cell. In yeast, expansion of the ER is observed under unfolded protein response (UPR) and subsequently induces autophagy initiated from the ER. Here, we found that soluble low molecular weight of Abeta disrupted the anchoring between ER and microtubules (MT) and induced collapse of ER. In addition, it decreased the stability of MT. Subsequently, low molecular weight Abeta triggered autophagy and enhanced lysosomal degradation, as shown by electron microscopy and live-cell imaging. Dysfunction of ER can be further proved in postmortem AD brain and transgenic mice bearing APP Swedish mutation by immunohistochemical analysis of calreticulin. Treatment with Taxol, a MT-stabilizing agent, could partially inhibit collapse of the ER and induction of autophagy. The results show that Abeta-induced disruption of MT can affect the architecture of the ER. Collapse/aggregation of the ER may play an important role in Abeta peptide-triggered neurodegenerative processes.

Polysaccharides from Wolfberry Antagonizes Glutamate Excitotoxicity in Rat Cortical Neurons

Glutamate excitotoxicity is involved in many neurodegenerative diseases including Alzheimer's disease (AD). Attenuation of glutamate toxicity is one of the therapeutic strategies for AD. Wolfberry (Lycium barbarum) is a common ingredient in oriental cuisines. A number of studies suggest that wolfberry has anti-aging properties. In recent years, there is a trend of using dried Wolfberry as food supplement and health product in UK and North America. Previously, we have demonstrated that a fraction of polysaccharide from Wolfberry (LBA) provided remarkable neuroprotective effects against beta-amyloid peptide-induced cytotoxicity in primary cultures of rat cortical neurons. To investigate whether LBA can protect neurons from other pathological factors such as glutamate found in Alzheimer brain, we examined whether it can prevent neurotoxicity elicited by glutamate in primary cultured neurons. The glutamate-induced cell death as detected by lactate dehydrogenase assay and caspase-3-like activity assay was significantly reduced by LBA at concentrations ranging from 10 to 500 microg/ml. Protective effects of LBA were comparable to memantine, a non-competitive NMDA receptor antagonist. LBA provided neuroprotection even 1 h after exposure to glutamate. In addition to glutamate, LBA attenuated N-methyl-D-aspartate (NMDA)-induced neuronal damage. To further explore whether LBA might function as antioxidant, we used hydrogen peroxide (H(2)O(2)) as oxidative stress inducer in this study. LBA could not attenuate the toxicity of H(2)O(2). Furthermore, LBA did not attenuate glutamate-induced oxidation by using NBT assay. Western blot analysis indicated that glutamate-induced phosphorylation of c-jun N-terminal kinase (JNK) was reduced by treatment with LBA. Taken together, LBA exerted significant neuroprotective effects on cultured cortical neurons exposed to glutamate.

Calcium Dysregulation in Alzheimer's Disease: from Mechanisms to Therapeutic Opportunities

Calcium is involved in many facets of neuronal physiology, including activity, growth and differentiation, synaptic plasticity, and learning and memory, as well as pathophysiology, including necrosis, apoptosis, and degeneration. Though disturbances in calcium homeostasis in cells from Alzheimer's disease (AD) patients have been observed for many years, much more attention was focused on amyloid-beta (Abeta) and tau as key causative factors for the disease. Nevertheless, increasing lines of evidence have recently reported that calcium dysregulation plays a central role in AD pathogenesis. Systemic calcium changes accompany almost the whole brain pathology process that is observed in AD, including synaptic dysfunction, mitochondrial dysfunction, presenilins mutation, Abeta production and Tau phosphorylation. Given the early and ubiquitous involvement of calcium dysregulation in AD pathogenesis, it logically presents a variety of potential therapeutic targets for AD prevention and treatment, such as calcium channels in the plasma membrane, calcium channels in the endoplasmic reticulum membrane, Abeta-formed calcium channels, calcium-related proteins. The review aims to provide an overview of the current understanding of the molecular mechanisms involved in calcium dysregulation in AD, and an insight on how to exploit calcium regulation as therapeutic opportunities in AD.

Modulation of Microglia by Wolfberry on the Survival of Retinal Ganglion Cells in a Rat Ocular Hypertension Model

The active component of Wolfberry (Lycium barbarum), lycium barbarum polysaccharides (LBP), has been shown to be neuroprotective to retinal ganglion cells (RGCs) against ocular hypertension (OH). Aiming to study whether this neuroprotection is mediated via modulating immune cells in the retina, we used multiphoton confocal microscopy to investigate morphological changes of microglia in whole-mounted retinas. Retinas under OH displayed slightly activated microglia. One to 100 mg/kg LBP exerted the best neuroprotection and elicited moderately activated microglia in the inner retina with ramified appearance but thicker and focally enlarged processes. Intravitreous injection of lipopolysaccharide decreased the survival of RGCs at 4 weeks, and the activated microglia exhibited amoeboid appearance as fully activated phenotype. When activation of microglia was attenuated by intravitreous injection of macrophage/microglia inhibitory factor, protective effect of 10 mg/kg LBP was attenuated. The results implicated that neuroprotective effects of LBP were partly due to modulating the activation of microglia.

Could PKR Inhibition Modulate Human Neurodegeneration?

Modulation of Neuroimmune Responses on Glia in the Central Nervous System: Implication in Therapeutic Intervention Against Neuroinflammation

It has long been known that the brain is an immunologically privileged site in normal conditions. Although the cascade of immune responses can occur as long as there is a neuronal injury or a potent immune stimulation, how the brain keeps glial cells in a quiescent state is still unclear. Increasing efforts have been made by several laboratories to elucidate how repression of immune responses is achieved in the neuronal environment. The suppression factors include neurotransmitters, neurohormones, neurotrophic factors, anti-inflammatory factors, and cell-cell contact via adhesion molecules or CD200 receptor. This review discusses how these factors affect the cascade of cerebral immune responses because no single factor listed above can fully account for the immune suppression. While several factors contribute to the suppression of immune responses, activation of glial cells and their production of pro-inflammatory factors do occur as long as there is a neuronal injury, suggesting that some neuronal components facilitate immune responses. This review also discusses which signals initiate or augment cerebral immune responses so that stimulatory signals override the suppressive signals. Increasing lines of evidence have demonstrated that immune responses in the brain are not always detrimental to neurons. Attempt to simply clear off inflammatory factors in the CNS may not be appropriate for neurons in neurological disorders. Appropriate control of immune cells in the CNS may be beneficial to neurons or even neuroregeneration. Therefore, understanding the mechanisms underlying immune suppression may help us to reshape pharmacological interventions against inflammation in many neurological disorders.

Erratum: Modulation of Microglia by Wolfberry on the Survival of Retinal Ganglion Cells in a Rat Ocular Hypertension Model

The active component of Wolfberry (Lycium barbarum), lycium barbarum polysaccharides (LBP), has been shown to be neuroprotective to retinal ganglion cells (RGCs) against ocular hypertension (OH). Aiming to study whether this neuroprotection is mediated via modulating immune cells in the retina, we used multiphoton confocal microscopy to investigate morphological changes of microglia in whole-mounted retinas. Retinas under OH displayed slightly activated microglia. One to 100 mg/kg LBP exerted the best neuroprotection and elicited moderately activated microglia in the inner retina with ramified appearance but thicker and focally enlarged processes. Intravitreous injection of bacterial endotoxin lipopolysaccharide (LPS) decreased the survival of RGCs at 4 weeks, and the activated microglia exhibited amoeboid appearance as fully activated phenotype. When activation of microglia was attenuated by intravitreous injection of macrophage/microglia inhibitory factor, protective effect of 10 mg/kg LBP was attenuated. The results implicated that neuroprotective effects of LBP were partly due to modulating the activation of microglia.[This corrects the article on p. in vol. .].

Protective Effects of Pinostilbene, a Resveratrol Methylated Derivative, Against 6-hydroxydopamine-induced Neurotoxicity in SH-SY5Y Cells

Resveratrol (3,4',5-trans-trihydroxystilbene) is a phytoalexin with emerging lines of evidence supporting its beneficial effects on cardiovascular systems and inhibition of carcinogenesis. It has also been reported that certain methylated resveratrol derivatives are more effective than resveratrol in the prevention/treatment of cancer. However, little is known about the impact of resveratrol and its derivatives on the development of Parkinson's disease. In this study, we compared the neuroprotective effects of resveratrol with four methylated (fully or partially) resveratrol derivatives against parkinsonian mimetic 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y cells. Release of lactate dehydrogenase and activity of caspase-3 triggered by 6-OHDA were significantly reduced by resveratrol and one of the methylated derivatives, pinostilbene (3,4'-dihydroxy-5-methoxystilbene), in a dose-dependent manner. In addition, pinostilbene exerted a potent neuroprotective effect with a wider effective concentration range than resveratrol. By using high-performance liquid chromatography, we found that uptake of pinostilbene into SH-SY5Y cells was significantly higher than that of resveratrol. Enhanced bioavailability may thus be a major factor contributing to the neuroprotective activity of pinostilbene. Moreover, Western blot analysis demonstrated that pinostilbene markedly attenuated the phosphorylation of JNK and c-Jun triggered by 6-OHDA. Besides, mammalian target of rapamycin kinase may be an intracellular target accounting for the neuroprotective effects of pinostilbene. Our findings demonstrate the potential of methylated stilbenes in neuroprotection and provide important information for further research in this field.

Anti-aging Herbal Medicine--how and Why Can They Be Used in Aging-associated Neurodegenerative Diseases?

Aging is a universal biological process that leads to progressive and deleterious changes in organisms. From ancient time, mankind has already interested in preventing and keeping ourselves young. Anti-aging study is certainly not a new research area. Nowadays, the meaning of anti-aging has been changed from simply prolonging lifespan to increasing health span, which emphasizes more on the quality of life. This is the concept of healthy aging and prevention of pathological aging, which is associated with diseases. Keeping our brain functions as in young age is an important task for neuroscientists to prevent aging-associated neurological disorders, such as Alzheimer's diseases (AD) and Parkinson's disease (PD). The causes of these diseases are not fully understood, but it is believed that these diseases are affected by multiple factors. Neurodegenerative diseases can be cross-linked with a number of aging-associated conditions. Based on this, a holistic approach in anti-aging research seems to be more reasonable. Herbal medicine has a long history in Asian countries. It is believed that many of the medicinal herbs have anti-aging properties. Recent studies have shown that some medicinal herbs are effective in intervention or prevention of aging-associated neurological disorders. In this review, we use wolfberry and ginseng as examples to elaborate the properties of anti-aging herbs. The characteristics of medicinal herbs, especially their applications in different disease stages (prevention and intervention) and multi-targets properties, allow them to be potential anti-aging intervention in prevention and treatment of the aging-associated neurological disorders.

A Pro-drug of the Green Tea Polyphenol (-)-epigallocatechin-3-gallate (EGCG) Prevents Differentiated SH-SY5Y Cells from Toxicity Induced by 6-hydroxydopamine

Regular consumption of green tea benefits people in prevention from cardiovascular disorders, obesity as well as neurodegenerative diseases. (-)-Epigallocatechin-3-gallate (EGCG) is regarded as the most biologically active catechin in green tea. However, the stability and bioavailability of EGCG are restricted. The purpose of the present study was to investigate whether a pro-drug, a fully acetylated EGCG (pEGCG), could be more effective in neuroprotection in Parkinsonism mimic cellular model. Retinoic acid (RA)-differentiated neuroblastoma SH-SY5Y cells were pre-treated with different concentrations of EGCG and pEGCG for 30 min and followed by incubation of 25 microM 6-hydroxydopamine (6-OHDA) for 24h. We found that a broad dosage range of pEGCG (from 0.1 to 10 microM) could significantly reduce lactate dehydrogenase release. Likewise, 10 microM of pEGCG was effective in reducing caspase-3 activity, while EGCG at all concentrations tested in the model failed to attenuate caspase-3 activity induced by 6-OHDA. Furthermore, Western-blot analysis showed that Akt could be one of the specific signaling pathways stimulated by pEGCG in neuroprotection. It was demonstrated that 25 microM of 6-OHDA significantly suppressed the phosphorylation level of Akt. Only pEGCG at 10 microM markedly increased its phosphorylation level compared to 6-OHDA alone. Taken together, as pEGCG has higher stability and bioavailability for further investigation, it could be a potential neuroprotective agent and our current findings may offer certain clues for optimizing its application in future.

Modulation of Morphological Changes of Microglia and Neuroprotection by Monocyte Chemoattractant Protein-1 in Experimental Glaucoma

Monocyte chemoattractant protein-1 (MCP-1)/CCL2 is a C-C chemokine involved in the activation and recruitment of monocytic cells to injury sites. MCP-1/CCL2 can induce either neuroprotection or neurodestruction in vitro, depending on the experimental model. We aim to use MCP-1/CCL2 as an experimental tool to investigate the morphological changes of microglia when loss of healthy retinal ganglion cells (RGCs) is exacerbated or attenuated in an experimental glaucoma model. While a high concentration (1000 ng) of MCP-1/CCL2 and lipopolysaccharide (LPS)-exacerbated RGC loss, 100 ng MCP-1/CCL2 provided neuroprotection towards RGC. Neuroprotective MCP-1/CCL2 (100 ng) also upregulated insulin-like growth factor-1 (IGF-1) immunoreactivity in the RGCs. The neuroprotective effect of MCP-1/CCL2 was not due to the massive infiltration of microglia/macrophages. Taken together, this is the first report showing that an appropriate amount of MCP-1/CCL2 can protect RGCs in experimental glaucoma.

Transcriptional Regulation of Human FE65, a Ligand of Alzheimer's Disease Amyloid Precursor Protein, by Sp1

FE65 is a neuronal-enriched adaptor protein that binds to the Alzheimer's disease amyloid precursor protein (APP). FE65 forms a transcriptionally active complex with the APP intracellular domain (AICD). The precise gene targets for this complex are unclear but several Alzheimer's disease-linked genes have been proposed. Additionally, evidence suggests that FE65 influences APP metabolism. The mechanism by which FE65 expression is regulated is as yet unknown. To gain insight into the regulatory mechanism, we cloned a 1.6 kb fragment upstream of the human FE65 gene and found that it possesses particularly strong promoter activity in neurones. To delineate essential regions in the human FE65 promoter, a series of deletion mutants were generated. The minimal FE65 promoter was located between -100 and +5, which contains a functional Sp1 site. Overexpression of the transcription factor Sp1 potentiates the FE65 promoter activity. Conversely, suppression of the FE65 promoter was observed in cells either treated with an Sp1 inhibitor or in which Sp1 was knocked down. Furthermore, reduced levels of Sp1 resulted in downregulation of endogenous FE65 mRNA and protein. These findings reveal that Sp1 plays a crucial role in transcriptional control of the human FE65 gene.

Neuroprotective Effects of Polysaccharides from Wolfberry, the Fruits of Lycium Barbarum, Against Homocysteine-induced Toxicity in Rat Cortical Neurons

Previous clinical and epidemiological studies have suggested that elevated plasma homocysteine (Hcy) levels increased the risk of Alzheime's disease (AD). Although the underlying mechanisms of its toxicity are elusive, it has been shown that Hcy damages neurons by inducing apoptosis, DNA fragmentation, and tau hyperphosphorylation. Wolfberry (Lycium barbarum) is a fruit that is known for its eye-protective and anti-aging properties in Asian countries. Previous studies from our laboratory have demonstrated that polysaccharides derived from wolfberry (LBA) have the ability to protect neurons from amyloid-beta (Abeta) peptide neurotoxicity. We hypothesize that the neuroprotective effects of wolfberry is not limited to Abeta and can also provide protection against other AD risk factors. In this study, we aim to elucidate the neuroprotective effects of wolfberry against Hcy-induced neuronal damage. Our data showed that LBA treatment significantly attenuated Hcy-induced neuronal cell death and apoptosis in primary cortical neurons as demonstrated by LDH and caspase-3 like activity assay. LBA also significantly reduced Hcy-induced tau phosphorylation at tau-1 (Ser198/199/202), pS396 (Ser396), and pS214 (Ser214) epitopes as well as cleavage of tau. At the same time, we also found that the phosphorylation level of p-GSK3beta (Ser9/Tyr 216) remained unchanged among different treatment groups at all detected time points. LBA treatment suppressed elevation of both p-ERK and p-JNK. In summary, our data demonstrated that LBA exerted neuroprotective effects on cortical neurons exposed to Hcy. Therefore, LBA has the potential to be a diseasemodifying agent for the prevention of AD.

Up-regulation of Crystallins is Involved in the Neuroprotective Effect of Wolfberry on Survival of Retinal Ganglion Cells in Rat Ocular Hypertension Model

Wolfberry (fruit of Lycium barbarum Linn) has been known for balancing 'Yin' and 'Yang' in the body, nourishing the liver and kidney, improving visual acuity for more than 2,500 years in oriental countries. The active components in wolfberry include L. barbarum polysaccharide (LBP), zeaxanthine, betaine, cerebroside and trace amounts of zinc, iron, and copper. Each of them confers distinct beneficial effects and together they help to explain widespread use of wolfberry in the eastern world. Earlier study reported the neuroprotective effects of LBP on retinal ganglion cell (RGC) in an experimental model of glaucoma and the underlying in vivo cellular mechanisms of LBP neuroprotection deserve further exploration. In this study, we adopted proteomics, functional genomics, to evaluate pharmacological effects of LBP on the neuronal survival pathways. Among the significantly changed proteins induced by LBP feeding on ocular hypertension (OH) retinas, only proteins in crystallin family were focused in this study. The proteomic results were further confirmed using the Western blotting of the retinas and immunohistochemical staining of the retinal sections. We demonstrated that neuroprotective effect of-wolfberry extract-LBP on the survival of RGCs may be mediated via direct up-regulation of neuronal survival signal betaB2-crystallin.

The Putative Neurodegenerative Links Between Depression and Alzheimer's Disease

Alzheimer's disease (AD) is the leading neurodegenerative cause of dementia in the elderly. Thus far, there is no curative treatment for this devastating condition, thereby creating significant social and medical burdens. AD is characterized by progressive cognitive decline along with various neuropsychiatric symptoms, including depression and psychosis. Depression is a common psychiatric disorder affecting individuals across the life span. Although the "monoamine hypothesis" of depression has long been proposed, the pathologies and mechanisms for depressive disorders remain only partially understood. Pharmacotherapies targeting the monoaminergic pathways have been the mainstay in treating depression. Additional therapeutic approaches focusing other pathological mechanisms of depression are currently being explored. Interestingly, a number of proposed mechanisms for depression appear to be similar to those implicated in neurodegenerative diseases, including AD. For example, diminishing neurotrophic factors and neuroinflammation observed in depression are found to be associated with the development of AD. This article first provides a concise review of AD and depression, then discusses the putative links between the two neuropsychiatric conditions.

Beneficial Effects of Cinnamon Proanthocyanidins on the Formation of Specific Advanced Glycation Endproducts and Methylglyoxal-induced Impairment on Glucose Consumption

Advanced glycation endproducts (AGEs) are a group of complex and heterogeneous compounds formed from nonenzymatic reactions. The accumulation of AGEs in vivo has been implicated as a major pathogenic process in diabetic complications and other health disorders, such as atherosclerosis and Alzheimer's disease, and normal aging. In this study, we investigate the inhibitory effects of cinnamon bark proanthocyanidins, catechin, epicatechin, and procyanidin B2 on the formation of specific AGE representatives including pentosidine, N(epsilon)-(carboxymethyl)lysine (CML), and methylglyoxal (MGO) derived AGEs. These compounds displayed obvious inhibitory effects on these specific AGEs, which are largely attributed to both their antioxidant activities and carbonyl scavenging capacities. Meanwhile, in terms of their potent MGO scavenging capacities, effects of these proanthocyanidins on insulin signaling pathways interfered by MGO were evaluated in 3T3-L1 adipocytes. According to the results, proanthocyanidins exerted protective effects on glucose consumption impaired by MGO in 3T3-L1 fat cells.

Modulation of Mitochondrial Calcium As a Pharmacological Target for Alzheimer's Disease

Perturbed neuronal calcium homeostasis is a prominent feature in Alzheimer's disease (AD). Mitochondria accumulate calcium ions (Ca(2+)) for cellular bioenergetic metabolism and suppression of mitochondrial motility within the cell. Excessive Ca(2+) uptake into mitochondria often leads to mitochondrial membrane permeabilization and induction of apoptosis. Ca(2+) is an interesting second messenger which can initiate both cellular life and death pathways in mitochondria. This review critically discusses the potential of manipulating mitochondrial Ca(2+) concentrations as a novel therapeutic opportunity for treating AD. This review also highlights the neuroprotective role of a number of currently available agents that modulate different mitochondrial Ca(2+) transport pathways. It is reasoned that these mitochondrial Ca(2+) modulators are most effective in combination with agents that increase the Ca(2+) buffering capacity of mitochondria. Modulation of mitochondrial Ca(2+) handling is a potential pharmacological target for future development of AD treatments.

Genetic Sphingosine Kinase 1 Deficiency Significantly Decreases Synovial Inflammation and Joint Erosions in Murine TNF-alpha-induced Arthritis

Sphingosine kinase 1 (SphK1) is an enzyme that converts sphingosine to bioactive sphingosine-1-phosphate. Recent in vitro data suggest a potential role of SphK1 in TNF-alpha-mediated inflammation. Our aims in this study were to determine the in vivo significance of SphK1 in TNF-alpha-mediated chronic inflammation and to define which pathogenic mechanisms induced by TNF-alpha are SphK1 dependent. To pursue these aims, we studied the effect of SphK1 deficiency in an in vivo model of TNF-alpha-induced chronic inflammatory arthritis. Transgenic hTNF-alpha mice, which develop spontaneous inflammatory erosive arthritis beginning at 14-16 wk, were crossed with SphK1 null mice (SphK1(-/-)), on the C57BL6 genetic background. Beginning at 4 mo of age, hTNF/SphK1(-/-) mice had significantly less severe clinically evident paw swelling and deformity, less synovial and periarticular inflammation, and markedly decreased bone erosions as measured quantitatively through micro-CT images. Mechanistically, the mice lacking SphK1 had less articular cyclooxygenase 2 protein and fewer synovial Th17 cells than did hTNF/SphK1(+/+) littermates. Microarray analysis and real-time RT-PCR of the ankle synovial tissue demonstrated that hTNF/SphK1(-/-) mice had increased transcript levels of suppressor of cytokine signaling 3 compared with hTNF/SphK1(+/+) mice, likely also contributing to the decreased inflammation in the SphK1-deficient mice. Finally, significantly fewer mature osteoclasts were detected in the ankle joints of hTNF/SphK1(-/-) mice compared with hTNF/SphK1(+/+) mice. These data indicate that SphK1 plays a key role in hTNF-alpha-induced inflammatory arthritis via impacting synovial inflammation and osteoclast number.

Sulfur-containing Constituents and One 1H-pyrrole-2-carboxylic Acid Derivative from Pineapple [Ananas Comosus (L.) Merr.] Fruit

Two sulfur-containing compounds, (S)-2-amino-5-((R)-1-carboxy-2-((E)-3-(4-hydroxy-3-methoxyphenyl)allylthio)ethyl-amino)-5-oxopentanoic acid (1) and (S)-2-amino-5-((R)-1-(carboxymethylamino)-3-((E)-3-(4-hydroxyphenyl)allylthio)-1-oxopropan-2-ylamino)-5-oxopentanoic acid (2), and one 1H-pyrrole-2-carboxylic acid derivative, 6-(3-(1H-pyrrole-2-carbonyloxy)-2-hydroxypropoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-carboxylic acid (3), together with eighteen known phenolic compounds, were isolated from the fruits of pineapple. Their structures were elucidated by a combination of spectroscopic analyses. Some of these compounds showed inhibitory activities against tyrosinase. The half maximal inhibitory concentration values of compounds 1, 4, 5, 6, 7 are lower than 1 mM. These compounds may contribute to the well-known anti-browning effect of pineapple juice and be potential skin whitening agents in cosmetic applications.

Temporal Relationship of Autophagy and Apoptosis in Neurons Challenged by Low Molecular Weight β-amyloid Peptide

Alzheimer's disease (AD) is an aging-related progressive neurodegenerative disorder. Previous studies suggested that various soluble Aβ species are neurotoxic and able to activate apoptosis and autophagy, the type I and type II programmed cell death, respectively. However, the sequential and functional relationships between these two cellular events remain elusive. Here we report that low molecular weight Aβ triggered cleavage of caspase 3 and poly (ADP-ribose) polymerase to cause neuronal apoptosis in rat cortical neurons. On the other hand, Aβ activated autophagy by inducing autophagic vesicle formation and autophagy related gene 12 (ATG12), and up-regulated the lysoso-mal machinery for the degradation of autophagosomes. Moreover, we demonstrated that activation of autophagy by Aβ preceded that of apoptosis, with death associated protein kinase phosphorylation as the potential molecular link. More importantly, under Aβ toxicity, neurons exhibiting high level of autophagosome formation were absent of apoptotic features, and inhibition of autophagy by 3-methylade-nine advanced neuronal apoptosis, suggesting that autophagy can protect neurons from Aβ-induced apoptosis.

Light Deprivation Induces Depression-like Behavior and Suppresses Neurogenesis in Diurnal Mongolian Gerbil (Meriones Unguiculatus)

Recent evidence suggests that adult neurogenesis contributes to the pathophysiology of different psychiatric disorders, including depressive disorder, anxiety disorder, and schizophrenia. Seasonal affective disorder (SAD) is a specific form of recurrent depressive disorder that can be induced by shortened light period. It is unclear yet whether neurogenesis is affected in SAD or under altered light/dark cycle. The present study aims at examining whether neurogenesis and dendritic growth of immature neurons are affected in Mongolian gerbils, a mainly diurnal rodent, under light deprivation. Animals were divided into two groups: the control (kept in 12 h light:12 h dark) and the light-deprived groups (kept in 24 h dark). Depression-like behaviors and neurogenesis were assessed after 2 weeks. Compared with the control group, light-deprived gerbils showed increased immobile time in the tail suspension test and forced swimming test, which indicates induction of depression-like behavior. Cell proliferation in both the hippocampal and subventricular zone were significantly decreased in the light-deprived group, which also showed a decreased neuronal differentiation. Dendritic maturation of immature neurons was suppressed by light deprivation, which is revealed by doublecortin staining and Sholl analysis. The results revealed that the light/dark cycle exerts impacts on neurogenesis and maturation of new neurons. Additionally, the current experiment may offer a model for exploring the relationship among daylight exposure, circadian cycles, depressive behavior, and the underlying mechanisms.

Lycium Barbarum Polysaccharides Reduce Neuronal Damage, Blood-retinal Barrier Disruption and Oxidative Stress in Retinal Ischemia/reperfusion Injury

Neuronal cell death, glial cell activation, retinal swelling and oxidative injury are complications in retinal ischemia/reperfusion (I/R) injuries. Lycium barbarum polysaccharides (LBP), extracts from the wolfberries, are good for "eye health" according to Chinese medicine. The aim of our present study is to explore the use of LBP in retinal I/R injury. Retinal I/R injury was induced by surgical occlusion of the internal carotid artery. Prior to induction of ischemia, mice were treated orally with either vehicle (PBS) or LBP (1 mg/kg) once a day for 1 week. Paraffin-embedded retinal sections were prepared. Viable cells were counted; apoptosis was assessed using TUNEL assay. Expression levels of glial fibrillary acidic protein (GFAP), aquaporin-4 (AQP4), poly(ADP-ribose) (PAR) and nitrotyrosine (NT) were investigated by immunohistochemistry. The integrity of blood-retinal barrier (BRB) was examined by IgG extravasations. Apoptosis and decreased viable cell count were found in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the vehicle-treated I/R retina. Additionally, increased retinal thickness, GFAP activation, AQP4 up-regulation, IgG extravasations and PAR expression levels were observed in the vehicle-treated I/R retina. Many of these changes were diminished or abolished in the LBP-treated I/R retina. Pre-treatment with LBP for 1 week effectively protected the retina from neuronal death, apoptosis, glial cell activation, aquaporin water channel up-regulation, disruption of BRB and oxidative stress. The present study suggests that LBP may have a neuroprotective role to play in ocular diseases for which I/R is a feature.

Drug Discovery from Chinese Medicine Against Neurodegeneration in Alzheimer's and Vascular Dementia

Alzheimer's disease and vascular dementia are two major diseases associated with dementia, which is common among the elderly. While the etiology of dementia is multi-factorial and complex, neurodegeneration may be the major cause of these two diseases. Effective drugs for treating dementia are still to be discovered. Current western pharmacological approaches against neurodegeneration in dementia develop symptom-relieving and disease-modifying drugs. Current integrative and holistic approaches of Chinese medicine to discovering drugs for neurodegeneration in dementia include (1) single molecules from the herbs, (2) standardized extracts from a single herb, and (3) herbal formula with definite composition. This article not only reviews the concept of dementia in western medicine and Chinese medicine but also evaluates the advantages and disadvantages of these approaches.

Neurodegeneration of the Retina in Mouse Models of Alzheimer's Disease: What Can We Learn from the Retina?

Alzheimer's disease (AD) is an age-related progressive neurodegenerative disease commonly found among elderly. In addition to cognitive and behavioral deficits, vision abnormalities are prevalent in AD patients. Recent studies investigating retinal changes in AD double-transgenic mice have shown altered processing of amyloid precursor protein and accumulation of β-amyloid peptides in neurons of retinal ganglion cell layer (RGCL) and inner nuclear layer (INL). Apoptotic cells were also detected in the RGCL. Thus, the pathophysiological changes of retinas in AD patients are possibly resembled by AD transgenic models. The retina is a simple model of the brain in the sense that some pathological changes and therapeutic strategies from the retina may be observed or applicable to the brain. Furthermore, it is also possible to advance our understanding of pathological mechanisms in other retinal degenerative diseases. Therefore, studying AD-related retinal degeneration is a promising way for the investigation on (1) AD pathologies and therapies that would eventually benefit the brain and (2) cellular mechanisms in other retinal degenerations such as glaucoma and age-related macular degeneration. This review will highlight the efforts on retinal degenerative research using AD transgenic mouse models.

Endoplasmic Reticulum Stress Induces Tau Pathology and Forms a Vicious Cycle: Implication in Alzheimer's Disease Pathogenesis

Accumulation of unfolded proteins can disturb the functions of the endoplasmic reticulum (ER), leading to ER-stress or unfolded protein response (UPR). Recent data have shown that activation of UPR can be found in postmortem brains of Alzheimer's disease (AD) patients; and biological markers for activation of UPR are abundant in neurons with diffuse phosphorylated tau. Although these observations suggest a linkage between ER-stress and tau pathology, little is known of their relationship. In this study, we found that high levels of phosphorylated PKR-like ER-resident kinase (p-PERK) and phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2α) as markers for activation of UPR in the hippocampus of aged P301L mutant tau transgenic mice. The immunoreactivity of p-PERK was found to co-localize with that of phosphorylated tau. We then hypothesized that phosphorylation of tau could induce ER-stress and vice versa in promoting AD-like pathogenesis. By using the protein phosphatase 2A inhibitor okadaic acid (OA) as an inducer for phosphorylation of tau, we found that primary cultures of rat cortical neurons treated with OA triggered UPR as indicated by increased levels of p-PERK and p-eIF2α, splicing of mRNA for xbp-1 and elevated levels of mRNA for GADD153. On the other hand, thapsigargin as an ER-stress inducer stimulated phosphorylation of tau at Thr231, Ser262 and Ser396. Thapsigargin also induced activation of caspase-3 and cleavage of tau. These findings suggested that ER-stress and hyperphosphorylation of tau could be induced by each other to form a vicious cycle to propagate AD-like neurodegeneration.

Lycium Barbarum Polysaccharides Protect Mice Liver from Carbon Tetrachloride-induced Oxidative Stress and Necroinflammation

Lycium barbarum has been used as a traditional Chinese medicine to nourish liver, kidneys and the eyes.

From Small to Big Molecules: How Do We Prevent and Delay the Progression of Age-related Neurodegeneration?

Age-related neurodegeneration in the brain and retina is complicated. It comprises a series of events encompassing different modes of degeneration in neurons, as well as inflammation mediated by glial cells. Systemic inflammation and risk factors can contribute to disease progression. Age-related conditions such as Alzheimer's disease (AD), Parkinson's disease (PD) and Age-related Macular Degeneration (AMD) affect patients for 5 to 20 years and are highly associated with risk factors such as hyperhomocysteinaemia, hypercholesterolaemia, hypertension, and symptoms of mood disorder. The long duration of the degeneration and the wide array of systemic factors provide the opportunity for nutraceutical intervention to prevent or delay disease progression. Small molecules such as phenolic compounds are candidates for neuroprotection because they have anti-oxidant activities and can modulate intracellular signaling pathways. Bigger entities such as oligosaccharides and polysaccharides have often been neglected because of their complex structure. However, certain big molecules can provide neuroprotective effects. They may also have a wide spectrum of action against risk factors. In this review we use an integrative approach to the potential uses of nutraceutical products to prevent age-related neurodegeneration. These include direct effects of phenolic compounds and polysaccharides on neurons to antagonize various neurodegenerative mechanisms in AD, PD and AMD, and indirect effects of these compounds on peripheral disease-related risk factors.

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