bluuebunny/crossref_metadata_2025_split huggingface dataset:
The nerve growth factor precursor proNGF exhibits neurotrophic activity but is less active than mature nerve growth factor
Authors: Fahnestock Margaret , Yu Guanhua , Michalski Bernadeta , Mathew Silvy , Colquhoun Amy , Ross Gregory M. , Coughlin Michael D. 
Published: December 2005

AbstractNerve growth factor (NGF) promotes neuronal survival and differentiation and stimulates neurite outgrowth. NGF is synthesized as a precursor, proNGF, which undergoes post‐translational processing to generate mature β‐NGF. It has been assumed that, in vivo, NGF is largely processed into the mature form and that mature NGF accounts for the biological activity. However, we recently showed that proNGF is abundant in CNS tissues whereas mature NGF is undetectable, suggesting that proNGF has biological functions beyond its role as a precursor. To determine whether proNGF exhibits biological activity, we mutagenized the precursor‐processing site and expressed unprocessed, cleavage‐resistant proNGF protein in insect cells. Survival and neurite outgrowth assays on murine superior cervical ganglion neurons and PC12 cells indicated that proNGF exhibits neurotrophic activity similar to mature 2.5S NGF, but is approximately fivefold less active. ProNGF binds to the high‐affinity receptor, TrkA, as determined by cross‐linking to PC12 cells, and is also slightly less active than mature NGF in promoting phosphorylation of TrkA and its downstream signaling effectors, Erk1/2, in PC12 and NIH3T3‐TrkA cells. These data, coupled with our previous report that proNGF is the major form of NGF in the CNS, suggest that proNGF could be responsible for much of the biological activity normally attributed to mature NGF in vivo.
17beta‐estradiol protects oligodendrocytes from cytotoxicity induced cell death
Authors: Takao Toshihiro , Flint Nicole , Lee Lan , Ying Xiaoyou , Merrill Jean , Chandross Karen J. 
Published: December 2005

AbstractDuring pregnancy, changes in circulating levels of hormones, including estrogens, correlates with a significant decrease in the relapse incidence in women with Multiple Sclerosis (MS). In the present study, we demonstrate that both primary and cell line cultures of rat oligodendrocytes express the estrogen receptor (ER)‐α and ERβ estrogen receptors in the cytosol and nucleus, and that nuclear compartmentalization becomes more pronounced as the cells mature. Moreover, 17β‐estradiol significantly decreases the cytotoxic effects of the peroxynitrite generator 3‐(4‐morpholinyl)‐sydnonimine (SIN‐1) in both immature and mature oligodendrocytes in a dose dependent manner. This protective mechanism requires pretreatment with 17β‐estradiol and is blocked by ICI 182,780, a selective ERα/ERβ antagonist. These results strongly suggest that 17β‐estradiol protects oligodendrocytes against SIN‐1 mediated cytotoxicity through the activation of the estrogen receptors and provides new insights into the roles of the estrogen signaling pathways in myelin forming cells that are lost in demyelinating disorders.
4‐Aminopyridine‐induced epileptogenesis depends on activation of mitogen‐activated protein kinase ERK
Authors: Merlo Daniela , Cifelli Pierangelo , Cicconi Simona , Tancredi Virginia , Avoli Massimo 
Published: December 2005

AbstractExtracellular signal‐regulated kinases such as ERK1 [p44 mitogen‐activated protein kinase (MAPK)] and ERK2 (p42 MAPK) are activated in the CNS under physiological and pathological conditions such as ischemia and epilepsy. Here, we studied the activation state of ERK1/2 in rat hippocampal slices during application of the K+channel blocker 4‐aminopyridine (4AP, 50 µm), a procedure that enhances synaptic transmission and leads to the appearance of epileptiform activity. Hippocampal slices superfused with 4AP‐containing medium exhibited a marked activation of ERK1/2 phosphorylation that peaked within about 20 min. These effects were not accompanied by changes in the activation state of c‐Jun N‐terminal kinase (JNK), another member of the MAP kinase superfamily. 4AP‐induced ERK1/2 activation was inhibited by the voltage‐gated Na+channel blocker tetrodotoxin (1 µm). We also found that application of the ERK pathway inhibitors U0126 (50 µm) or PD98059 (100 µm) markedly reduced 4AP‐induced epileptiform synchronization, thus abolishing ictal discharges in the CA3 area. The effects induced by U0126 or PD98059 were not associated with changes in the amplitude and latency of the field potentials recorded in the CA3 area following electrical stimuli delivered in the dentate hylus. These data demonstrate that activation of ERK1/2 accompanies the appearance of epileptiform activity induced by 4AP and suggest a cause‐effect relationship between the ERK pathway and epileptiform synchronization.
Comparison of in vivo and in vitro subcellular localization of estrogen receptors α and β in oligodendrocytes
Authors: Zhang Zhan , Cerghet Mirela , Mullins Chadwick , Williamson Maryann , Bessert Denise , Skoff Robert 
Published: December 2005

AbstractThe existence of estrogen receptors (ERs) in oligodendrocytes (OLGs) in vivo and in vitro is unresolved, as their presence has been reported in some studies and their absence in others. Using molecular and immunocytochemical techniques, we describe the subcellular localization of ERα and ERβ in OLGs in vivo and in vitro. Both ERα and ERβ are detected in an immortalized OLG cell line and in enriched OLG cultures by RT‐PCR and western blot. Immunocytochemistry of OLGs from enriched cultures shows ERα receptors are nuclear, whereas ERβ receptors are cytoplasmic. Confocal and deconvolution microscopy of enriched OLG cultures reveals ERβ immunoreactivity is concentrated in perikarya and veins of OLG membrane sheets; lesser reactivity is present in their plasma membranes and nuclei. In vivo, we readily detect ERα in neurons but not in OLGs, even though we used different fixation procedures and different ERα antibodies. The presence of ERα in cultured OLGs may be due to culture media that contains factors stimulating ERα expression but are reduced in normal brain. In vivo, ERβ immunoreactivity is readily detectable in OLG cytoplasm and in myelin sheaths. Incubation of glial cultures without or with increasing concentrations of 17β–estradiol (E2) shows that E2 significantly accelerates OLG process formation.
Dopamine transporter‐mediated cytotoxicity of β‐carbolinium derivatives related to Parkinson's disease: relationship to transporter‐dependent uptake
Authors: Storch Alexander , Hwang Yu‐I , Gearhart Debra A. , Beach J. Warren , Neafsey Edward J. , Collins Michael A. , Schwarz Johannes 
Published: December 2005

AbstractEndogenous or exogenous β‐carboline (βC) derivatives structurally related to the selective dopaminergic neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and its active metabolite 1‐methyl‐4‐phenylpyridinium (MPP+) may contribute to dopaminergic neurodegeneration in Parkinson's disease (PD). We addressed the importance of the dopamine transporter (DAT) for selective dopaminergic toxicity by testing the differential cytotoxicity and cellular uptake of 12 βCs in human embryonic kidney HEK‐293 cells ectopically expressing the DAT gene. Cell death was measured using [4,5‐Dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide (MTT) and trypan blue exclusion assays, and uptake by a fluorescence‐based uptake assay. All βCs and MPP+ showed general cytotoxicity in parental HEK‐293 cells after 72 h with half‐maximal toxic concentrations (TC50 values) in the upper micromolar range. Besides MPP+, only 2[N]‐methylated compounds showed enhanced cytotoxicity in DAT expressing HEK‐293 cells with 1.3‐ to 4.5‐fold reduction of TC50 values compared with parental cell line. The rank order of selectivity was: MPP+ >> 2[N],9[N]‐dimethyl‐harminium > 2[N]‐methyl‐harminium > 2[N],9[N]‐dimethyl‐harmanium = 2[N]‐methyl‐norharmanium > 2[N]‐methyl‐harmanium > 2[N],9[N]‐dimethyl‐norharminium. Consistently, only 2[N]‐methylated βCs were transported into the cell through the DAT with up to five times greater Km and 12–220 times smaller Vmax values compared with dopamine and MPP+. There was a weak relation of DAT‐mediated selectivity with the affinity of βCs at the DAT (Km), but not with Vmax. Our data suggest that DAT‐mediated cellular uptake of 2[N]‐methylated βCs represents a potential mechanism for selective toxicity towards dopaminergic neurons and may be relevant for the pathogenesis of Parkinson's disease.
Interaction between dopamine and its transporter: role of intracellular sodium ions and membrane potential
Authors: Chen Nianhang , Reith Maarten E. A. 
Published: December 2005

AbstractThe present study addresses the effect of intracellular Na+and membrane potential on the binding of dopamine (DA) to the dopamine transporter (DAT). Perforation of plasma membranes of DAT‐expressing cells with gramicidin diminished DA uptake and decreased the potency (increases Ki) of DA in inhibiting the binding of cocaine analog [3H]2β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane (CFT). It also compromised the ability of external Na+to reduce DA Ki. No substantial effect on DA Kiwas observed upon gramicidin treatment in Na+‐free buffer, membrane depolarization with high [K+]o, or elevation of [Na+]iwith monensin under non‐depolarizing conditions. Elevation of DA Kiwas greater at more positive potentials when [Na+]iwas raised to a similar level, or at higher [Na+]iwhen the membrane was depolarized to a similar level. In cells expressing D313N DAT, DA Kiwas significantly higher but less sensitive to gramicidin than that in wild‐type (WT) cells. In contrast, DA Kiin cell‐free membranes was insensitive to Na+, gramicidin, and D313N mutation. The data suggest that (i) intracellular Na+plays a role in affecting the external access to DA binding sites at DAT on depolarized plasma membranes of cells, and (ii) access to DA binding sites in cell‐free membranes may occur from the intracellular side of the membrane. Unlike DA binding, CFT binding to both cells and membranes was sensitive to Na+and D313N mutation but insensitive to gramicidin, consistent with exclusively external access to sites that are different from but conformationally linked to those for DA.
Effects of 6‐hydroxydopamine on primary cultures of substantia nigra: specific damage to dopamine neurons and the impact of glial cell line‐derived neurotrophic factor
Authors: Ding Yun Min , Jaumotte Juliann D. , Signore Armando P. , Zigmond Michael J. 
Published: December 2005

Abstract6‐Hydroxydopamine (6‐OHDA)‐induced loss of dopamine (DA) neurons has served to produce an animal model of DA neuron loss in Parkinson's disease. We report here the use of 6‐OHDA to produce an in vitro model of this phenomena using dissociated cultures prepared from neonatal rat mesencephalon. Cultures were exposed to 6‐OHDA (40–100 μm, 15 min) in an antioxidant medium, and DA and GABA neurons evaluated by immunocytochemistry. 6‐OHDA induced morphological and biochemical signs of cell death in DA neurons within 3 h, followed by loss of tyrosine hydroxylase immunoreactive neurons within 2 days. In substantia nigra (SN) cultures, DA neurons were much more affected by 6‐OHDA than were GABA neurons. In contrast, DA neurons from the ventral tegmental area were only lost at higher, non‐specific concentrations of 6‐OHDA. The effects of 6‐OHDA on nigral DA neurons were blocked by inhibitors of high affinity DA transport and by z‐DEVD‐fmk (150 μm), a caspase inhibitor. Glial cell line‐derived neurotrophic factor (GDNF) treatment reduced TUNEL labeling 3 h after 6‐OHDA exposure, but did not prevent loss of DA neurons at 48 h. Thus, 6‐OHDA can selectively destroy DA neurons in post‐natal cultures of SN, acting at least in part by initiating caspase‐dependent apoptosis, and this effect can be attenuated early but not late by GDNF.
Cellular localization and functional expression of P‐glycoprotein in rat astrocyte cultures
Authors: Ronaldson Patrick T. , Bendayan Moise , Gingras Diane , Piquette‐Miller Micheline , Bendayan Reina 
Published: December 2005

AbstractWe investigated the cellular/subcellular localization and functional expression of P‐glycoprotein, an ATP‐dependent membrane‐associated efflux transporter, in astrocytes, a brain parenchyma compartment that is poorly characterized for the expression of membrane drug transporters. Analyses were carried out on primary cultures of astrocytes isolated from the cerebral cortex of neonatal Wistar rats and CTX TNA2, an immortalized rat astrocyte cell line. Both cell cultures display morphological features typical of type I astrocytes. RT‐PCR analysis revealed mdr1a and mdr1b mRNA in primary cultures of astrocytes and in CTX TNA2 cells. Western blot analysis using the P‐glycoprotein monoclonal C219 antibody detected a single band of appropriate size in both cell systems. Immunocytochemical analysis using the monoclonal antibodies C219 and MRK16 labeled P‐glycoprotein along the plasma membrane, caveolae, coated vesicles and nuclear envelope. Immunoprecipitation studies using the caveolin‐1 polyclonal H‐97 antibody demonstrated that P‐glycoprotein is physically associated with caveolin‐1 in both cell culture systems. The accumulation of [3H]digoxin (an established P‐glycoprotein substrate) by the astrocyte cultures was significantly enhanced in the presence of standard P‐glycoprotein inhibitors and an ATP depleting agent. These results demonstrate the cellular/subcellular location and functional expression of P‐glycoprotein in rat astrocytes and suggest that this glial compartment may play an important role in the regulation of drug transport in the CNS.
Theory for the impact of basal turnover on dopamine clearance kinetics in the rat striatum after medial forebrain bundle stimulation and pressure ejection
Authors: Michael Adrian C. , Borland Laura M. , J. Mitala Joseph , Willoughby Bridget M. , Motzko Christina M. 
Published: December 2005

AbstractAlthough microdialysis measurements suggest that extracellular dopamine concentrations in the rat striatum are in the low nanomolar range, some recent voltammetry studies suggest that the concentration may be considerably higher, perhaps in the micromolar range. The presence of such high dopamine levels in the extracellular space has to be rationalized with the rapid, linear clearance of extracellular dopamine observed after electrical stimulation of the medial forebrain bundle. Kinetic analysis of dopamine clearance after evoked release suggests that the basal extracellular dopamine concentration is below theKMof dopamine uptake, which is near 0.2 µm. However, dopamine clearance after pressure ejection of dopamine into the rat striatum is slow and non‐linear, which may alternatively be a sign that basal dopamine release is only slightly slower than the maximal velocity of dopamine uptake,Vmax. A high basal extracellular dopamine concentration would exist if basal dopamine release were only slightly slower than theVmaxof uptake. This report introduces a new kinetic analysis of dopamine uptake that sheds light on the possible source of the different clearance rates observed following evoked dopamine release and dopamine pressure ejection. Furthermore, the analysis rationalizes the rapid dopamine clearance after evoked release with the possibility that basal extracellular dopamine levels are above theKMof the transporter.
Familial Alzheimer's disease mutations inhibit γ‐secretase‐mediated liberation of β‐amyloid precursor protein carboxy‐terminal fragment
Authors: Wiley Jesse C. , Hudson Mark , Kanning Kevin C. , Schecterson Leslyanne C. , Bothwell Mark 
Published: December 2005

AbstractCleavage of the β‐secretase processed β‐amyloid precursor protein by γ‐secretase leads to the extracellular release of Aβ42, the more amyloidogenic form of the β‐amyloid peptide, which subsequently forms the amyloid‐plaques diagnostic of Alzheimer's disease. Mutations in β‐amyloid precursor protein (APP), presenilin‐1 and presenilin‐2 associated with familial Alzheimer's disease (FAD) increase release of Aβ42, suggesting that FAD may directly result from increased γ‐secretase activity. Here, we show that familial Alzheimer's disease mutations clustered near the sites of γ‐secretase cleavage actually decrease γ‐secretase‐mediated release of the intracellular fragment of APP (CTFγ). Concordantly, presenilin‐1 mutations that result in Alzheimer's disease also decrease the release of CTFγ. Mutagenesis of the epsilon cleavage site in APP mimicked the effects of the FAD mutations, both decreasing CTFγ release and increasing Aβ42 production, suggesting that perturbation of this site may account for the observed decrement in γ‐secretase‐mediated proteolysis of APP. As CTFγ has been implicated in transcriptional activation, these data indicate that decreased signaling and transcriptional regulation resulting from FAD mutations in β‐amyloid precursor protein and presenilin‐1 may contribute to the pathology of Alzheimer's disease.
Acute anoxia induces tau dephosphorylation in rat brain slices and its possible underlying mechanisms
Authors: Liu Rong , Pei Jin‐Jing , Wang Xiao‐Chuan , Zhou Xin‐Wen , Tian Qing , Winblad Bengt , Wang Jian‐Zhi 
Published: December 2005

AbstractAbnormal phosphorylation of microtubule‐associated protein tau plays a critical role in Alzheimer's disease (AD), together with a distinct decrease of energy metabolism in the affected brain regions. To explore the effect of acute energy crisis on tau phosphorylation and the underlying mechanisms, we incubated rat brain slices in artificial cerebrospinal fluid (aCSF) at 37°C with or without an oxygen supply, or in aCSF with low glucose concentrations. Then, the levels of total, phosphorylated and unphosphorylated tau, as well as the activities and levels of protein phosphatase (PP)‐1, PP‐2A, glycogen synthase kinase 3 (GSK‐3), extracellular signal‐regulated protein kinase (ERK) and C‐jun amino terminal kinase (JNK), were measured. It was found, unexpectedly, that tau was significantly dephosphorylated at Ser396/Ser404 (PHF‐1), Ser422 (R145), Ser199/Ser202 (Tau‐1), Thr181 (AT270), Ser202/Thr205 (AT8) and Thr231 (AT180) by acute anoxia for 30 min or 120 min. The activity of PP‐2A and the level of dephosphorylated PP‐2A catalytic subunit at tyrosine 307 (Tyr307) were simultaneously increased. The active forms of ERK1/2 and JNK1/2 were decreased under anoxic incubation. The PP‐2A inhibitor, okadaic acid (OA, 0.75 µm), completely prevented tau from acute anoxia‐induced dephosphorylation and restored the active forms of ERK1/2 and JNK1/2 to the control level. The activities and protein levels of GSK‐3 and PP‐1 showed no change during acute anoxia. These data suggest that acute anoxia induces tau dephosphorylation, and that PP‐2A may play a key role in tau dephosphorylation induced by acute anoxia.
In vivo evidence of CHIP up‐regulation attenuating tau aggregation
Authors: Sahara Naruhiko , Murayama Miyuki , Mizoroki Tatsuya , Urushitani Makoto , Imai Yuzuru , Takahashi Ryosuke , Murata Shigeo , Tanaka Keiji , Takashima Akihiko 
Published: December 2005

AbstractThe carboxyl terminus of heat‐shock cognate (Hsc)70‐interacting protein (CHIP) is a ubiquitin E3 ligase that can collaborate with molecular chaperones to facilitate protein folding and prevent protein aggregation. Previous studies showed that, together with heat‐shock protein (Hsp)70, CHIP can regulate tau ubiquitination and degradation in a cell culture system. Ubiquitinated tau is one component in neurofibrillary tangles (NFTs), which are a major histopathological feature of Alzheimer's disease (AD). However, the precise sequence of events leading to NFT formation and the mechanisms involved remain unclear. To confirm CHIP's role in suppressing NFT formation in vivo, we performed a quantitative analysis of CHIP in human and mouse brains. We found increased levels of CHIP and Hsp70 in AD compared with normal controls. CHIP levels in both AD and controls corresponded directly to Hsp90 levels, but not to Hsp70 or Hsc70 levels. In AD samples, CHIP was inversely proportional to sarkosyl‐insoluble tau accumulation. In a JNPL3 mouse brain tauopathy model, CHIP was widely distributed but weakly expressed in spinal cord, which was the most prominent region for tau inclusions and neuronal loss. Protein levels of CHIP in cerebellar regions of JNPL3 mice were significantly higher than in non‐transgenic littermates. Human tau was more highly expressed in this region of mouse brains, but only moderate levels of sarkosyl‐insoluble tau were detected. This was confirmed when increased insoluble tau accumulation was found in mice lacking CHIP. These findings suggest that increases in CHIP may protect against NFT formation in the early stages of AD. If confirmed, this would indicate that the quality‐control machinery in a neuron might play an important role in retarding the pathogenesis of tauopathies.
Differences in gene expression profile among SH‐SY5Y neuroblastoma subclones with different neurite outgrowth responses to nerve growth factor
Authors: Oe Tomoya , Sasayama Takeshi , Nagashima Takeyuki , Muramoto Masakazu , Yamazaki Takao , Morikawa Noriyuki , Okitsu Osamau , Nishimura Shintaro , Aoki Toshiaki , Katayama Yoshiki , Kita Yasuhiro 
Published: December 2005

AbstractNerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF‐induced neurite‐positive (NIN+) subclones that showed high responsiveness to NGF‐induced neurite outgrowth and three NGF‐induced neurite‐negative (NIN–) subclones that abolished NGF‐induced neurite outgrowth from parental SH‐SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506‐mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF‐induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF‐induced increases in mRNAs for the immediate‐early genes (IEGs) c‐fosand NGF inducible (NGFI) genesNGFI‐A,NGFI‐BandNGFI‐C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN– cell line, particularly in terms of axonal vesicle‐related genes and growth cone guidance‐related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN– cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH‐SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.