Distinct effects of atypical 1,4-dihydropyridines on 1-methyl-4-phenylpyridinium-induced toxicity

Our previous data obtained from in vivo experiments demonstrated high neuroprotective effects of three novel atypical neuronal non-calcium antagonistic 1,4-dihydropyridine (DHP) derivatives cerebrocrast, glutapyrone and tauropyrone. The present studies were carried out in vitro to clarify, at least in part, their mechanism of action in primary culture of cerebellar granule cells by use of 1-methyl-4-phenylpyridinium (MPP+) as a neurotoxic agent which causes dramatic oxidative stress. Cerebrocrast (highly lipophilic, with a classical two-ring structure) dose-dependently (0.01-10.0 microM, EC50 = 13 nM) reduced MPP+-induced cell death. At the same time, the calcium antagonist nimodipine (reference drug) protected cell death at much higher concentrations (EC50 = 12.4 microM). Cerebrocrast decreased also the generation of reactive oxygen species and loss of mitochondrial membrane potential. In contrast, low lipophilic amino acid-containing DHPs glutapyrone and tauropyrone (glutamate- and taurine-containing, correspondingly) were without significant effects indicating their distinct mode of action in comparison to cerebrocrast. We have demonstrated for the first time an ability of atypical non-calcium antagonistic DHP cerebrocrast (which has classical DHP structure elements and high lipophilicity) to protect MPP+-induced deterioration of mitochondrial bioenergetics. One may suggest mitochondria as an essential intracellular target for the neuroprotective action of cerebrocrast and indicate its usefulness in the treatment of Parkinson's disease.     

Dietary variation and stress among prehistoric Jomon foragers from Japan

Current archaeological evidence indicates that greater dietary reliance on marine resources is recorded among the eastern Jomon, while plant dependence prevailed in western/inland Japan. The hypothesis that the dietary choices of the western/inland Jomon will be associated with greater systemic stress is tested by comparing carious tooth and enamel hypoplasia frequencies between the eastern and western/inland Jomon. Demographic collapse coincides with climate change during the Middle to Late Jomon period, suggesting dwindling resource availability. It is hypothesized that this change was associated with greater systemic stress and/or dietary change among the Middle to Late Jomon. This hypothesis is tested by comparing enamel hypoplasia and carious tooth frequencies between Middle to Late and Late to Final Jomon foragers. Enamel hypoplasia was significantly more prevalent among the western/inland Jomon. Such findings are consistent with archaeological studies that argue for greater plant consumption and stresses associated with seasonal resource depletion among the western/inland Jomon. Approximately equivalent enamel hypoplasia frequencies between Middle to Late and Late to Final Jomon foragers argues against a demographic collapse in association with diminished nutritional returns. Significant differences in carious tooth frequencies are, however, observed between Middle to Late and Late to Final Jomon foragers. These results suggest a subsistence shift during the Middle to Late Jomon period, perhaps in response to a changed climate. The overall patterns of stress documented by this study indicate wide-spread environmentally directed biological variation among the prehistoric Jomon.     

Alteration of cytosolic free calcium homeostasis by SIN-1: high sensitivity of L-type Ca2+ channels to extracellular oxidative/nitrosative stress in cerebellar granule cells

Exposure of cerebellar granule neurones in 25 mm KCl HEPES-containing Locke's buffer (pH 7.4) to 50-100 microm SIN-1 during 2 h decreased the steady-state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 +/- 33 nm to 60 +/- 10 nm, whereas exposure to > or = 0.3 mm SIN-1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 +/- 10 nm (due to inactivation of L-type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN-1-induced necrotic cell death. Both effects of SIN-1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4-benzoic acid)porphyrin chloride. Supplementation of Locke's buffer with catalase before addition of 0.5-1 mm SIN-1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60-120 min exposure to SIN-1 and also protected against SIN-1-induced necrotic cell death. alpha-Tocopherol, the potent NMDA receptor antagonist (+)-MK-801 and the N- and P-type Ca2+ channels blocker omega-conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN-1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60-120 min exposure to 0.5-1 mm SIN-1. It is concluded that L-type Ca2+ channels are a primary target of SIN-1-induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5-1 microm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.     

Extracellular release of newly synthesized sphingosine-1-phosphate by cerebellar granule cells and astrocytes

Sphingosine-1-phosphate (S1P) is a potent biomediator that can act as either an intracellular or an intercellular messenger. In the nervous system it exerts a wide range of actions, and specific membrane receptors for it have been identified in various regions. However, the physiological origin of extracellular S1P in the nervous system is largely unknown. We investigated cerebellar granule cells at different stages of differentiation and astrocytes in primary cultures as possible origins of extracellular S1P. Although these cells show marked differences in S1P metabolism, we found that they can all release S1P and express mRNAs for S1P specific receptors. Extracellular S1P derives from the export of newly synthesized intracellular S1P, and not from the action of a released sphingosine kinase. S1P release is rapid, efficient, and can be regulated by exogenous stimuli. Phorbol ester treatment resulted in an increase in sphingosine kinase 1 activity in the membranes, accompanied by a significant increase in extracellular S1P. S1P release in cells from the cerebellum emerges as a regulated mechanism, possibly related to a specific pool of newly synthesized S1P. To our knowledge, this is the first evidence of the extracellular release of S1P by primary cells from the CNS, which supports a role of S1P as autocrine/paracrine physiological messenger in the cerebellum.     

Inhibition of neuronal apoptosis by the cyclin-dependent kinase inhibitor GW8510: identification of 3' substituted indolones as a scaffold for the development of neuroprotective drugs

Increasing evidence suggests that neuronal apoptosis is triggered by the inappropriate activation of cyclin-dependent kinases leading to an abortive re-entry of neurons into the cell cycle. Pharmacological inhibitors of cell-cycle progression may therefore have value in the treatment of neurodegenerative diseases in humans. GW8510 is a 3' substituted indolone that was developed recently as an inhibitor of cyclin-dependent kinase 2 (CDK2). We found that GW8510 inhibits the death of cerebellar granule neurons caused by switching them from high potassium (HK) medium to low potassium (LK) medium. Although GW8510 inhibits CDK2 and other CDKs when tested in in vitro biochemical assays, when used on cultured neurons it only inhibits CDK5, a cytoplasmic CDK that is not associated with cell-cycle progression. Treatment of cultured HEK293T cells with GW8510 does not inhibit cell-cycle progression, consistent with its inability to inhibit mitotic CDKs in intact cells. Neuroprotection by GW8510 is independent of Akt and MEK-ERK signaling. Furthermore, GW8510 does not block the LK-induced activation of Gsk3beta and, while inhibiting c-jun phosphorylation, does not inhibit the increase in c-jun expression observed in apoptotic neurons. We also examined the effectiveness of other 3' substituted indolone compounds to protect against neuronal apoptosis. We found that like GW8510, the VEGF Receptor 2 Kinase Inhibitors [3-(1H-pyrrol-2-ylmethylene)-1,3-dihydroindol-2-one], {(Z)-3-[2,4-Dimethyl-3-(ethoxycarbonyl)pyrrol-5-yl)methylidenyl]indol-2-one} and [(Z)-5-Bromo-3-(4,5,6,6-tetrahydro-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one], the Src family kinase inhibitor SU6656 and a commercially available inactive structural analog of an RNA-dependent protein kinase inhibitor 5-Chloro-3-(3,5-dichloro-4-hydroxybenzylidene)-1,3-dihydro-indol-2-one, are all neuroprotective when tested on LK-treated neurons. Along with our recent identification of the c-Raf inhibitor GW5074 (also a 3' substituted indolone) as a neuroprotective compound, our findings identify the 3' substituted indolone as a core structure for the designing of neuroprotective drugs that may be used to treat neurodegenerative diseases in humans.     

Building and breeding molecules to spy on cells and tumors

Imaging of biochemical processes in living cells and organisms is essential for understanding how genes and gene products work together in space and time and in health and disease. Such imaging depends crucially on indicator molecules designed to maximize sensitivity and specificity. These molecules can be entirely synthetic, entirely genetically encoded macromolecules, or hybrid combinations, each approach having its own pros and cons. Recent examples from the author's laboratory include peptides whose uptake into cells is triggered by proteases typical of tumors, monomeric red fluorescent proteins and biarsenical-tetracysteine systems for determining the age and electron-microscopic location of proteins.     

Loss of βarrestin1 and βarrestin2 contributes to pulmonary hypoplasia and neonatal lethality in mice

Less is known about the connection between the malfunction of βarrestins and developmental defects as the mice with either of two βarrestin isoforms knockout appear normal. In order to address the biological function of βarrestins during developmental process, we generate βarrestin1/2 double knockout mice. We found that βarrestin1/2 dual-null mice developed respiratory distress and atelectasis that subsequently caused neonatal death. Morphological examination revealed type II pneumocyte immaturity. Our results indicate that not only βarrestin1/2 double knockout lung tissue show disturbances in cell proliferation but βarrestin1 and βarrestin2 contribute to pulmonary surfactant complex generation during pulmonary maturation. Intra-amniotic delivery of recombinant adenovirus expressing βarrestin1 or βarrestin2 enhances surfactant-associated proteins synthesis in vivo. Our mRNA microarray data further reveal that βarrestin1/2 double knockout results in downregulation of a significant proportion of genes involved in several lung morphogenesis processes. Together, our study demonstrates that βarrestin1 and βarrestin2 collaborate in embryonic development processes for epithelial pneumocyte differentiation and lung maturation.     

Age-related changes of structures in cerebellar cortex of cat

We studied the structures of the cerebellar cortex of young adult and old cats for age-related changes, which were statistically analysed. Nissl staining was used to visualize the cortical neurons. The immunohistochemical method was used to display glial fibrillary acidic protein (GFAP)-immunoreactive (IR) astrocytes and neurofilament-immunoreactive (NF-IR) neurons. Under the microscope, the thickness of the cerebellar cortex was measured; and the density of neurons in all the layers as well as that of GFAP-IR cells in the granular layer was analysed. Compared with young adult cats, the thickness of the molecular layer and total cerebellar cortex was significantly decreased in old cats, and that of the granular layer increased. The density of neurons in each layer was significantly lower in old cats than in young adult ones. Astrocytes in old cats were significantly denser than in young adult ones, and accompanied by evident hypertrophy of the cell bodies and enhanced immunoreaction of GFAP substance. Purkinje cells (PCs) in old cats showed much fewer NF-IR dendrites than those in young adults. The above findings indicate a loss of neurons and decrease in the number of dendrites of the PCs in the aged cerebellar cortex, which might underlie the functional decline of afferent efficacy and information integration in the senescent cerebellum. An age-dependent enhancement of activity of the astrocytes may exert a protective effect on neurons in the aged cerebellum