bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation of aromatic l‐amino acid decarboxylase by calcium/calmodulin kinase II
Authors: Neff N. H. , Duchemin A‐M. , Hadjiconstantinou Maria 
Published: July 2010

Calcium/calmodulin‐dependent protein kinase II (CAMKII) is widely distributed in the brain, and is involved in the regulation of neurotransmitter synthesis. Tyrosine hydroxylase and tryptophan hydroxylase, the two enzymes involved in the first step in the synthesis of catecholamines and indolamines, respectively, are phosphorylated by CAMKII. We now found that aromatic l‐amino acid decarboxylase (AAAD), the common second enzyme of both synthetic pathways, is activated by CAMKII. AAAD activity was assayed in mouse striatum homogenates with L‐DOPA as a substrate. The activation of AAAD by CAMKII in vitro is time and concentration‐dependent with maximal activity observed with 1.5 μg/mL of purified brain CAMKII and a 20‐min incubation. CAMKII induces a 30% increase of the apparent Vmax of the enzyme without changing its affinity for L‐DOPA and the cofactor pyridoxal‐5′‐phosphate. We have previously shown that AAAD can be phosphorylated by cyclic AMP‐and cyclic GMP‐dependent protein kinases. These two kinases induced an increase of AAAD activity of about 20%. Phosphorylation by several differentially regulated serine‐threonine kinases might be the mechanism responsible for the short‐term kinetic activation of the enzyme, which has been found in vivo after physiological stimuli and neurotransmitter receptor modulation.Acknowledgements:  Supported, in part, by grant NS34571.
Lorenzo's oil, adrenoleukodystrophy, and the blood– brain barrier
Authors: Murphy E. J. 
Published: July 2010

Adrenoleukodystrophy is a rapid, progressive demyelinating disease affecting the CNS that is characterized by large increases in plasma and tissue very long saturated fatty acids (VLCFA). Lorenzo's oil (LO), consisting of erucic (22:1 n‐9) and oleic (18:1 n‐9) acid in a triglyceride form, is a dietary therapy effective in reducing plasma and tissue VLCFA. Despite the decreased VLCFA, clinical studies indicated that LO failed to stop the progressive demyelination, suggesting that erucic acid, the active component of LO, did not cross the BBB. We addressed this question by infusing [14‐14C] 22:1 n‐9 (170 μCi/kg) into male rats using two different infusion paradigms. The radiotracer was infused (i.v.) into awake, adult male rats over a 10‐min period or infused (i.c.v.) into the fourth ventricle over a 7‐day period using an osmotic mini‐pump. Brains were removed from the cranium, frozen in liquid nitrogen, lipids extracted, and separated using standard techniques. [1‐14C] 20:4 n‐6 was infused (i.v.) and used as a positive control. Following i.v. infusion, 0.011% of the erucic acid was extracted by the brain, compared to 0.055% of the arachidonic acid. About 60% of the brain erucic acid was found in the aqueous fraction compared to 30% for arachidonic acid. Further, erucic acid was targeted to cholesteryl ester and triacylglyceride pools, whereas arachidonic acid was targeted to phospholipid pools. In animals infused i.c.v., 0.078% of the dose was taken up and about 60% of the erucic acid was targeted to phospholipid pools. These results clearly demonstrate that erucic acid crosses the BBB, similar to arachidonic acid, and is incorporated into specific lipid pools.Acknowledgements:  This work was supported by The Myelin Project.
Hypothalamic ependymal cells express the glucose transporter GLUT2, a protein involved in glucose sensing
Authors: Nualart F. , García M. A. , Ulloa V. , Silva C. , Pastor P. , Vera J. C. , Reinicke K. 
Published: July 2010

The GLUT2 glucose transporter is expressed in pancreatic islet β cells participating in glucose sensing mechanisms. In addition to glucose, GLUT2 is required in the transport of fructuose. In situ hybridization analyses of GLUT2 gene expression suggested that this transporter is present in hypothalamic regions responsible for glucose sensing. The hypothalamic glial cells, known as α and β tanycytes, are specialized ependymal cells that bridge the CSF and the blood. We used immunocytochemistry and transport analyses to demonstrate that GLUT1 and GLUT2 are expressed in tanycytes and are directly involved in the uptake of glucose and fructose. We also demonstrated that GLUT1 expression is elevated in β‐1 tanycytes, as compared to neurons and astrocytes. In addition, confocal microscopy analysis using anti‐GLUT2 indicated that GLUT2 was specifically expressed in β‐1 and β‐2 tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was found to express both GLUT1 and GLUT2. Analyses of 2‐deoxy‐d‐glucose and fructose uptake in cultured tanycytes revealed the presence of two transporters with transport Kms and sensitivity to inhibitors consistent with the functional properties of GLUT1 and GLUT2. Therefore, we conclude that mouse hypothalamic tanycytes express functional GLUT1 and GLUT2. Because the expression of GLUT2 is linked to glucose sensing mechanisms, we postulate that glial cells participate, at least in part, in glucose sensing mechanisms.Acknowledgements:  Supported by Grands FONDECYT 1010843 and DIUC‐GIA 201.034.006–1.4 from Concepción University.
Gene expression for glycosyltransferases in transfected neuroblastoma cells
Authors: Irwin L. N. , Bieberich E. , Li D. D. , Silva J. , Yu R. K. 
Published: July 2010

To study regulatory mechanisms of ganglioside biosynthesis, genes for sialyltransferase II (ST2) or acetylgalactosaminyltransferase I (GalNAcT) were transfected into different cell lines of a substrain of murine neuroblastoma F‐11 cells (F‐11A). While complex β‐series ganglioside synthesis is negligible in the parental line, cells transfected with genes for either enzyme express complex gangliosides requiring both enzyme activities. We have used primers specific for each of the two genes to determine the level of gene expression (1) in the parental cells where endogenous ST2 and GalNAcT activities are very low, and (2) in each of the transfected cell lines where both enzyme activities are present and complex gangliosides are synthesized. Transcripts for both ST2 and GalNAcT were amplified from cDNA that was reverse‐transcribed from the parental as well as both transfected cell lines. While some differences in transcript number and size were noted between parental, ST2‐transfected, and GalNAcT‐transfected cells, suggesting some degree of transcriptional control, all the cell lines clearly expressed transcripts for both genes. These results support the view that ST2 and GalNAcT act in a coordinated fashion, perhaps as a multienzyme complex, to catalyze the synthesis of complex gangliosides, and that synthesis of complex β‐series gangliosides in these cells is regulated primarily post‐transcriptionally.Acknowledgements:  Supported by NIH R15‐DC05179 to L.N.I, NIH NS‐11853 to R.K.Y, and NIH‐NCRR(RCMI) G12‐RRO8124 to the Border Biomedical Research Center at the University of Texas at El Paso.
N‐acetyl cysteine protects against injury in animal model of transient focal cerebral ischemia
Authors: Sekhon B. K. , Sekhon C. S. , Khan M. , Barbosa E. , Patel S. J. , Singh I. , Singh A. K. 
Published: July 2010

Ischemic cerebrovascular disease (stroke) is one of the leading causes of death and long‐term disability worldwide (or developing countries). The purpose of this study is to investigate the therapeutic potential of N‐acetyl cysteine (NAC), a precursor of glutathione, and a potent antioxidant, in tissue protection against transient focal cerebral ischemia model in rats. Our laboratory has already demonstrated the efficacy of NAC against induction of proinflammatory cytokines, iNOS, and production of NO in cultured cells. In this study, 250–300 g male Sprague–Dawley rats were subjected to 45 min of reversible focal cerebral ischemia through middle cerebral artery (MCA) occlusion. The protective effect of NAC is supported by reduced infarct volume as well as the suppression of TNF‐α and iNOS in the treated animals. Pretreatment with NAC reduced the infarct size by 49.6% as compared to the saline treated group along with reducing the neurological evaluation score by more than 50%. Our results suggest that preadministration of NAC attenuates focal cerebral ischemia by protecting cells against free radical damage.Acknowledgements:  Supported by NIH grants NS‐40144, NS‐40810, NS‐22576, NS‐34741 and NS‐37766.
Up‐regulation of `peripheral‐type' benzodiazepine receptors in the globus pallidus in manganese neurotoxicity
Authors: Hazell A. S. , Normandin L. 
Published: July 2010

Increasing evidence suggests that astrocytes play a major role in damage to the nervous system in manganese neurotoxicity (MN). Astrocytes accumulate manganese and sequester the metal in mitochondria where it inhibits oxidative phosphorylation. Chronic exposure to manganese leads to selective neuronal loss in basal ganglia structures such as the globus pallidus together with characteristic astrocytic changes known as Alzheimer type II astrocytosis. In previous reports we have demonstrated that treatment of these cells with manganese leads to increased binding site densities of the ‘peripheral‐type’ benzodiazepine receptor (PTBR) complex (Hazell et al. 1999; Neurosci. Lett. 271, 5–8), found predominantly in astrocytes and an important source of neurosteroid production. In the present study, we have examined the effects of manganese treatment in a subacute rat model of manganese neurotoxicity. Male Sprague–Dawley rats (200 g) were administered manganese (II) chloride (50 mg/kg, i.p. in 0.2 mL saline) daily for 1 and 4 days while control animals were injected with carrier only. Using neutron activation analysis, levels of manganese in the globus pallidus were found to be increased by 81% after 24 h of treatment but were elevated by 551% compared to controls after 4 days. Manganese treatment also led to a 110% increase in levels of the 18 kDa isoquinoline carboxamide‐binding protein, a major constituent of the PTBR, after 4 days but with no change in protein expression at 1 day by immunoblotting, consistent with the neutron activation findings. These results indicate that PTBRs and possibly neurosteroids are an early response to manganese exposure and may play a major role in the pathophysiology of MN.
Zinc causes loss of membrane potential and alters production of reactive oxygen species in isolated brain mitochondria
Authors: Dineley K. E. , Richards L. L. , Votyakova T. V. , Reynolds I. J. 
Published: July 2010

Excessive accumulation of intracellular free zinc is lethal to neurons. The precise mechanism by which zinc kills neurons remains unclear, however, a number of reports suggest that zinc may impair neuronal metabolism. The present study examined how zinc affects the activity of isolated rat brain mitochondria. Using spectrofluorometry and polarography, we determined the effects of zinc on mitochondrial membrane potential (Δψ), reactive oxygen species (ROS) generation, and respiration. In mitochondria respiring on either glutamate + malate (G + M), succinate, or glycerol 3‐phosphate (G3P), addition of submicromolar levels of zinc caused dissipation of delta psi. Zinc‐induced depolarization was rapidly reversed by the addition of the membrane‐impermeant metal chelator EGTA. In all three substrate conditions, ruthenium red (200 nm) partially protected mitochondria from zinc‐induced depolarization, suggesting that the calcium uniporter may mediate mitochondrial zinc uptake. Conversely, addition of cyclosporin A (1 µM) afforded no protection, arguing against zinc‐induced permeability transition. ROS production was enhanced by zinc in mitochondria fueled by either G + M or G3P, but zinc inhibited succinate‐driven ROS production. Zinc inhibited respiration in phosphorylating mitochondria regardless of substrate conditions, and zinc also inhibited respiration in uncoupled mitochondria using G + M. Together, these results suggest that zinc has several deleterious effects on mitochondria that could contribute to neuronal injury.Acknowledgements:  Supported by NS34138 and the American Heart Association.
Glutamate‐mediated CA++ influx and nuclear damage in retinal ganglion cells purified by panning: role of glia
Authors: DeCoster Mark A. , Bazan Nicolas G. 
Published: July 2010

The visual acuity decline in glaucoma is due to degeneration of retinal ganglion cells. We used cell panning with the antibody Thy 1.1 to isolate retinal ganglion cells (RGCs) from rat retina. Individual RGCs were extremely sensitive to glutamate, demonstrating sustained calcium influx responses to 100 nm glutamate. We tested unoprostone for its ability to elicit neuroprotection. This docosanoid decreases intraocular pressure in glaucoma patients and protects mixed retinal ganglion cell cultures from glutamate excitotoxicity and calcium influx. In the presence of retinal glia, RGCs responded to 100 and 500 nm glutamate with transient calcium influx, demonstrating glial buffering effects. Pure retinal glial cultures required on average at least 1 mm glutamate to trigger calcium influx, which was transient and oscillating. Unoprostone (10 μm) inhibited calcium influx stimulated by 5 mm glutamate in retinal glial cultures, while DMSO did not. Unoprostone at 1 μm also blocked neuronal calcium responses to 100 nm glutamate in mixed cultures, while DMSO did not. In RGCs isolated by panning, unoprostone blocked calcium responses to 100 nm glutamate in some cells, while DMSO blocked none. Unoprostone also protected RGCs from 20 μm glutamate‐induced toxicity and nuclear damage. We hypothesize that while unoprostone may affect individual RGCs depending on a putative receptor, the presence of retinal glia also plays a key role in neuroprotection and unoprostone action. The use of the cellular model described here and whole retina preparations will be useful to understand the mechanisms of RGC survival.Acknowledgements:  Support: Novartis Ophthalmics, Basel, Switzerland.
Estrogen as a neuroprotective agent in rat spinal cord injury
Authors: Banik N. L. , Sribnick E. A. , Wingrave J. M. , Matzelle D. D. , Wilford G. G. , Ray S. K. 
Published: July 2010

Spinal cord injury (SCI) is a neurological problem affecting approximately 11 000 Americans each year. Several treatment agents have been proposed; however, only methylprednisolone has limited efficacy. Estrogen is a multiactive neuroprotectant with antioxidant and anti‐inflammatory properties and attenuates calcium (Ca2+) influx following neuronal injury. To examine the neuroprotective effects of estrogen in SCI, we induced SCI (40 g/cm injury) in rats. Treatment groups were sham (laminectomy only), SCI plus vehicle, and SCI plus estrogen. Injured rats were treated with either 4 mg/kg 17 β‐estradiol (estrogen group) or dimethylsulfoxide (vehicle group) at 15 min and 24 h following injury. All rats were killed at 48 h to analyze SCI segments for calpain content and Bax/Bcl‐2 ratio by Western blotting. Tissue was also examined using calcium green‐2 to measure intracellular [Ca2+], JC‐1 to measure mitochondrial membrane potential, and double immunofluorescence for macrophages and calpain. Calpain content in the lesion penumbra, adjacent to the injury, was higher in vehicle than sham and this increase was attenuated in estrogen treated rats. In the lesion penumbra, the Bax/Bcl‐2 ratio was increased in vehicle rats as compared to sham. This increase was attenuated in estrogen treated rats. Estrogen treated rats had less Ca2+ influx, less inflammatory cell infiltration, and increased maintenance of mitochondrial membrane potential compared to vehicle treated rats. Our preliminary data suggest that estrogen may be effective in decreasing Ca2+ influx, inflammatory cell infiltration, and Bax/Bcl‐2 ratio following SCI.Acknowledgements:  Supported in part by grants from NIH‐NINDS and South Carolina Electric and Gas.
Effect of neuronal NOS inhibitor, 7‐nitro‐indazole, on cytosolic caspase 9 activity in vitro
Authors: Mishra O. P. 
Published: July 2010

Previous studies have shown that nitric oxide synthase (NOS) inhibitors prevent or reduce the hypoxia‐induced neuronal injury. The present study tests the hypothesis that neuronal NOS inhibitor, 7‐nitro‐indazole (7‐NI) will inhibit the cysteine protease, caspase 9, the initiator of programmed cell death. The effect of 7 NI on caspase 9 activity was determined in the cytosolic fraction of the cerebral cortex of normoxic (Nx, n = 4) and hypoxic (Hx, n = 4) newborn piglets. The animals were exposed to either 21% (Nx) or 7% (Hx) oxygen for 60 min. Cerebral tissue hypoxia was documented by the ATP and phosphocreatine levels. Cytosols were prepared and caspase 9 activity determined using a specific fluorogenic substrate (‐Ac‐Leu‐Glu‐His‐Asp‐AFC) for caspase 9 and monitored at 400 nm excitation and 505 nm emission wavelength. Caspase 9 activity in Nx group was 629 ± 49 as compared to 960 ± 105 pmoles AFC/min/mg protein (p < 0.002, 50.2% increase) in the Hx group. In presence of 10, 50 and 100 μm 7‐NI, caspase 9 activity decreased to 459 ± 41, 252 ± 36 and 256 ± 29 pmoles/min/mg protein, respectively, in Nx group. In Hx group, the activity decreased to 578 ± 49, 402 ± 37, 393 ± 35 pmoles/min/mg protein, respectively. The data demonstrate that 7‐NI inhibits caspase 9 in both the Nx and Hx groups. It is concluded that 7‐NI is an inhibitor of caspase 9. It is proposed that 7 NI administration will be neuroprotective due to its properties as inhibitor of nNOS as well as of caspase 9 and prevent the hypoxia‐induced neuronal death.Acknowledgements:  Supported by NIH‐HD‐38079.
A novel oxidative stress dependent apoptotic pathway in pesticide‐induced dopaminergic degeneration in PD models
Authors: Kanthasamy A. , Kitazawa M. , Kaul S. , Anantharam S. V. , Kanthasamy A. G. 
Published: July 2010

Emerging epidemiological and case control studies suggest that environmental factors especially pesticides are dominant risk factors in the etiology of idiopathic Parkinson's disease (PD). In the present study, we characterized oxidative stress dependent cellular events by exposing rat mesencephalic dopaminergic cell line (N27) and PC 12 cells to dieldrin (a potential environmental risk factor for development of PD). Exposure of dieldrin (30–200 μm) resulted in a rapid increase in generation of reactive oxygen species within 30 min and was followed by release of mitochondrial cytochrome‐c into cytoplasm. Cytochrome‐c then activated a proapoptotic cysteine protease, caspase‐3 in a time‐ and dose‐dependent manner. Interestingly, we also found that dieldrin exposure induces a time‐ and dose‐dependent proteolytic cleavage of native protein kinase Cδ (PKCδ, 74 kDa) to yield 41‐kDa catalytically active and 38‐kDa regulatory fragments. Pretreatment with caspase inhibitors blocked dieldrin‐induced proteolytic cleavage of PKCδ, indicating that cleavage is mediated by caspase‐3. Attenuation of ROS generation, caspase‐3 activation or overexpression of mutant PKCδK376R (dominant negative) almost completely suppressed dieldrin‐induced DNA fragmentation. We also found that dieldrin exposure activates the redox sensitive transcription factor NF‐kB through PKCδ dependent manner. Together, these data demonstrate that caspase‐3 dependent proteolytic activation of PKCδ plays a key role in oxidative stress‐mediated apoptosis in dopaminergic cells following exposure to an environmental neurotoxic agent.Acknowledgements:  Supported in part by ES 10586.
Symposium 3: Function and Pathogenesis in Parkinson’s Disease. Synuclein: the third amyloid in Alzheimer's disease
Authors: Trojanowski J. Q. 
Published: July 2010

A dramatic paradigm shift in understanding Parkinson's disease (PD) has emerged with implications for Alzheimer's disease (AD) because: (1) Mutations in the alpha‐synuclein (AS) gene cause familial PD, (2) Antibodies to AS detect Lewy bodies (LBs) and dystrophic Lewy neurites in PD, dementia with LBs (DLB), sporadic AD and the LB variant of AD (LBVAD), (3) Insoluble AS filaments are recovered from DLB brains and purified LBs, (4) Recombinant AS assembles into LB‐like filaments and residues 71–82 are essential for filament assembly, (5) AS transgenic mice and flies develop a PD‐like phenotype, (6) Cortical LBs detected with antibodies to AS correlate with dementia in PD, DLB and LBVAD, (7) Antibodies to AS detect LBs in 50% of familial AD, sporadic AD and Down's syndrome brains, (8) AS forms glial cytoplasmic inclusions (GCIs) in multiple system atrophy, (9) Epitopes throughout AS in LBs and GCIs, (10) Filamentous AS aggregates in LBs, GCIs and related lesions contain nitrated tyrosines, (11) Cells transfected with AS and treated with nitric oxide generators develop LB‐like AS inclusions, (12) Bigenic mice overexpressing mutant human APP and AS show an augmentation in AS inclusions. Thus, neurodegenerative diseases characterized by AS pathologies are synucleinopathies, and the filamentous AS lesions in these disorders may result in part from oxidative/nitrative damage to AS. Abnormal interactions of brain proteins may underlie synucleinopathies and other neurodegenerative disorders. Acknowledgements:Supported by NIA/NIH and Alzheimer's Association.
GM1 ganglioside in the treatment of Parkinson's disease: long‐term clinical experience
Authors: Schneider J. S. 
Published: July 2010

The monosialoganglioside GM1 is a glycosphingolipid with putative neuroprotective and neurorestorative properties. GM1 has been shown to at least partially reverse neurochemical, neuropathological and behavioral deficits in various lesion models including experimental models of parkinsonism in rodents, felines and nonhuman primates. Based on these experimental findings, we conducted a randomized double blind placebo controlled trial of GM1 in Parkinson's disease (PD). The results of this study showed significant improvements in Unified Parkinson's Disease Rating Scale (UPDRS) motor ratings as well as in other indices of motor function and activities of daily living assessments in PD patients that received 16 weeks of GM1 treatment (Schneider et al. Neurology 1998; 50: 1630–1636). At the conclusion of that study, patients were allowed to receive GM1 in an open extension trial. Fifteen patients chose to receive GM1 and remained in the open trial for approximately 4 years; 11 patients completed the full 5 years open trial. Chronic daily use of GM1 ganglioside by Parkinson's disease patients, over a period of 4–5 years is safe and has led to significant and sustained improvements in motor functioning, in most cases. In addition, we have observed a lower than expected frequency of motor complications of levodopa therapy (i.e. levodopa‐induced dyskinesias) in this patient group. Although the precise mechanisms of action of GM1 ganglioside are still unclear, animal and in vitro studies suggest that GM1 may interfere with apoptotic and to a lesser extent, necrotic cell death and stimulate cell survival and perhaps fiber/terminal sprouting through a variety of cellular/molecular processes.