Differential Response of Neural Cells to Trauma-Induced Free Radical Production In Vitro

CNS trauma has been associated with an increase in free radical production, but the cellular sources of this increase or the mechanism involved in the production of free radicals are not known. We, therefore, investigated the effects of trauma on free radical production in cultured neurons, astrocytes and BV-2 microglial cells. Free radicals were measured with the fluorescent dye DCFDA following in vitro trauma. At 30 and 60 min following trauma, there was a 132% and 64% increase, respectively, in free radical production in neurons when compared to controls. In astrocytes, there was a 94% and 133% increase at 30 and 60 min, respectively. Microglial cells, however, displayed no significant increase in free radicals at 30, 60 or 120 min following trauma. Since trauma can induce the mitochondrial permeability transition (MPT), a process associated with mitochondrial dysfunction, we further investigated whether cyclosporin A (CsA), an agent known to block the MPT, could prevent free radical formation following trauma. In neurons CsA did not block free radical production at 30 min but blocked it by 90% at 60 min. In contrast, in astrocytes CsA completely blocked free radical production at 30 min but did not block it at 60 min. Our results indicate that a differential sensitivity to trauma-induced free radical production exists in neural cells; that the MPT may be involved in the production of free radical post-trauma; and that the CsA-sensitive phase of free radical production is different in neurons and astrocytes.     

Uptake of Mannose-Terminal Glucocerebrosidase in Cultured Human Cholinergic and Dopaminergic Neuron Cell Lines

Enzyme replacement therapy has been shown to be particularly effective for patients with type 1 (non-neuronopathic) Gaucher disease. However, intravenously administered glucocerebrosidase does not reverse or halt the progression of brain damage in patients with type 2 (acute neuronopathic) Gaucher disease. A previous investigation revealed that intracerebral infusion of mannose-terminal glucocerebrosidase was safe in experimental animals. The enzyme had a comparatively long half-life in the brain. It was transported by convection from the site of infusion along white matter fiber tracts to the cerebral cortex where it was endocytosed by neurons. In anticipation of intracerebral administration of mannose-terminal glucocerebrosidase to patients with type 2 Gaucher disease, it was important to learn the mechanism involved in its cellular uptake. We therefore compared the endocytosis of this enzyme by J774 macrophage cells with that in two human neuronal cell lines and a human astrocyte cell line. Mannose-terminal glucocerebrosidase was taken up by cholinergic LA-N-2 cells, but to a much lower extent than by macrophages. Considerably less of the enzyme was endocytosed by dopaminergic SH-SY5Y cells. It was not taken up by NHA astrocytes. The findings provide encouragement for an exploration of intracerebral administration of glucocerebrosidase in patients with type 2 Gaucher disease.     

Necdin interacts with the ribonucleoprotein hnRNP U in the nuclear matrix

Necdin is expressed predominantly in terminally differentiated neurons, and its ectopic expression suppresses cell proliferation. We screened a cDNA library from neurally differentiated embryonal carcinoma P19 cells for necdin-binding proteins by the yeast two-hybrid assay. One of the positive clones contained cDNA encoding a carboxyl-terminal portion of heterogeneous nuclear ribonucleoprotein U (hnRNP U), a nuclear matrix-associated protein that interacts with chromosomal DNA. We isolated cDNA encoding full-length mouse hnRNP U to analyze its physical and functional interactions with necdin. The necdin-binding site of hnRNP U was located near a carboxyl-terminal region that mediated the association between hnRNP U and the nuclear matrix. In postmitotic neurons, endogenously expressed necdin and hnRNP U were detected in the nuclear matrix and formed a stable complex. Ectopically expressed necdin was concentrated in the nucleoli, but coexpressed hnRNP U recruited necdin to the nucleoplasmic compartment of the nuclear matrix. Furthermore, under the same conditions necdin and hnRNP U cooperatively suppressed the colony formation of transfected SAOS-2 cells. These results suggest that necdin suppresses cell proliferation through its interaction with hnRNP U in the specific subnuclear structure.     

Colonic Mechanoreceptor Input to Neurons in Guinea Pig Caudal Mesenteric Ganglion

The background activity of pacemaker-like neurons (PLN) of the guinea-pig caudal mesenteric ganglion (CMG) and their reflex responses to colonic distention were studied on combined isolated preparations including the CMG and a colon segment connected with the lumbar colonic nerves. The results allow us to suggest that the spontaneous and reflex activity of PLN is of a peripheral origin. Synaptic transmission in the peripheral colonofugal nerve pathways is mediated by acetylcholine and substance P (SP). The SP-induced excitation of PLN probably involves activation of the tachykinin NK₃ receptors.     

An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells

Intracellular reactive iron is a source of free radicals and a possible cause of cell damage. In this study, we analyzed the changes in iron homeostasis generated by iron accumulation in neuroblastoma (N2A) cells and hippocampal neurons. Increasing concentrations of iron in the culture medium elicited increasing amounts of intracellular iron and of the reactive iron pool. The cells had both IRP1 and IRP2 activities, being IRP1 activity quantitatively predominant. When iron in the culture medium increased from 1 to 40 microm, IRP2 activity decreased to nil. In contrast, IRP1 activity decreased when iron increased up to 20 microm, and then, unexpectedly, increased. IRP1 activity at iron concentrations above 20 microm was functional as it correlated with increased (55) Fe uptake. The increase in IRP1 activity was mediated by oxidative-stress as it was largely abolished by N-acetyl-L-cysteine. Culturing cells with iron resulted in proteins and DNA modifications. In summary, iron uptake by N2A cells and hippocampus neurons did not shut off at high iron concentrations in the culture media. As a consequence, iron accumulated and generated oxidative damage. This behavior is probably a consequence of the paradoxical activation of IRP1 at high iron concentrations, a condition that may underlie some processes associated with neuronal degeneration and death.     

Cholesterol accumulates in cell bodies,but is decreased in distal axons,of Niemann-Pick C1-deficient neurons

Niemann-Pick type-C (NPC) disease is characterized by a progressive loss of neurons and an accumulation of unesterified cholesterol within the endocytic pathway. Unlike other tissues, however, NPC1-deficient brains do not accumulate cholesterol but whether or not NPC1-deficient neurons accumulate cholesterol is not clear. Therefore, as most studies on cholesterol homeostasis in NPC1-deficient cells have been performed in fibroblasts we have investigated cholesterol homeostasis in cultured murine sympathetic neurons lacking functional NPC1. These neurons did not display obvious abnormalities in growth or morphology and appeared to respond normally to nerve growth factor. Filipin staining revealed numerous cholesterol-filled endosomes/lysosomes in NPC1-deficient neurons and the mass of cholesterol in cell bodies was greater than in wild-type neurons. Surprisingly, however, the cholesterol content of NPC1-deficient and wild-type neurons as a whole was the same. This apparent paradox was resolved when the cholesterol content of NPC1-deficient distal axons was found to be less than of wild-type axons. Cholesterol sequestration in cell bodies did not depend on exogenously supplied cholesterol since the cholesterol accumulated before birth and did not disperse when neurons were cultured without exogenous cholesterol. The altered cholesterol distribution between cell bodies and axons suggests that transport of cholesterol, particularly that synthesized endogenously, from cell bodies to distal axons is impaired in NPC1-deficient neurons.     

Activation of Ca2+/calmodulin-dependent protein kinase I in cultured rat hippocampal neurons

We have focused on activation mechanisms of calcium/calmodulin-dependent protein kinase (CaM) kinase I in the hippocampal neurons and compared them with that of CaM kinase IV. Increased activation of CaM kinase I occurred by stimulation with glutamate and depolarization in cultured rat hippocampal neurons. Similar to CaM kinases II and IV, CaM kinase I was essentially activated by stimulation with the NMDA receptor. Although both CaM kinases I and IV seem to be activated by CaM kinase kinase, the activation of CaM kinase I was persistent during stimulation with glutamate in contrast to a transient activation of CaM kinase IV. In addition, CaM kinase I was activated in a lower concentration of glutamate than that of CaM kinase IV. Depolarization-induced activation of CaM kinase I was also evident in the cultured neurons and was largely blocked by nifedipine. In the experiment with 32P-labeled cells, phosphorylation of CaM kinase I was stimulated by glutamate treatment and depolarization. The glutamate- and depolarization-induced phosphorylation was inhibited by the NMDA receptor antagonist and nifedipine, respectively. These results suggest that, although CaM kinases I and IV are activated by the NMDA receptor and depolarization stimulation, these kinase activities are differently regulated in the hippocampal neurons.     

Excitatory action of lead on rat sympathetic preganglionic neurons in vitro and in vivo

Lead exposure elicited an increase in blood pressure and was considered to be a cardiovascular risk factor. The involvements of sympathetic nervous system and circulating catecholamines have been implicated in lead-induced hypertension. This study examined the effects of PbCl(2) on sympathetic preganglionic neurons (SPNs) in vitro and in vivo. In vitro electrophysiological study showed that superfusion of a low concentration (5 microM) of PbCl(2), which had no effects on membrane potential and spontaneous discharge rate, enhanced excitatory postsynaptic potentials (EPSPs) in some of the SPNs examined but inhibited inhibitory postsynaptic potentials (IPSPs) in other SPNs tested. A higher concentration (50 microM) of PbCl(2) inhibited both EPSPs and IPSPs in all SPNs examined. In vivo study showed that intrathecal injection of PbCl(2) (10 and 100 nmol) via an implanted cannula to the T7-T9 segments of urethane-anesthetized rats increased both the heart rate and mean arterial pressure. The pressor and tachycardic responses of intrathecal PbCl(2) (100 nmol) were attenuated by pretreatment with intravenous administration of hexamethonium (10 mg/kg) or intrathecal AP-5 (DL-2-amino-5-phosphonovaleric acid, 100 nmol), but were not significantly antagonized by prior intrathecal administration of CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, 100 nmol). Taken together, these results demonstrated that lead may exert a stimulatory effect on SPNs, which may result from the enhancement of EPSPs and inhibition of IPSPs by low concentrations of lead.     

Effects of GABA on circular smooth muscle spontaneous activities of rat distal colon

GABAergic regulation of intestinal motility through the modulation of non-adrenergic non-cholinergic (NANC) neurons remains poorly understood especially in rat colon where very few studies have been undertaken. Therefore, the effects of GABA on circular preparations of rat distal colon were investigated using classical organ bath chambers to record spontaneous mechanical activities (SMA). SMA was characterized by the occurrence of rhythmic phasic contractions (type-I) or by spontaneously occurring large contractions superimposed on small rhythmic contractions (type-II). In the presence of atropine and guanethidine (NANC conditions), these large contractions were inhibited by bicuculline, a GABA(A)-receptor antagonist as well as by TTX, L-NAME and apamin together, or L 732-138, a NK1-receptor antagonist. In NANC conditions, GABA induced a transient monophasic relaxation or a biphasic effect characterized by a relaxation followed by a tonic contraction in both type-I and -II preparations. Both the inhibitory and excitatory effects of GABA were blocked by TTX and L-NAME + apamin; the GABA-induced contraction was also sensitive to L 732-138. The responses to GABA were mimicked by the GABA(A)-receptor agonist, muscimol, whereas baclofen and CACA, respectively GABA(B) and GABA(C)-receptors agonists showed no effect. These results demonstrated that only GABA(A)-receptors seem to be involved in the regulation of SMA in rat distal colon in NANC conditions. Release of NANC inhibitory transmitter (NO and probably ATP) and NANC excitatory transmitter (maybe substance P) might be involved.     

Necdin is required for terminal differentiation and survival of primary dorsal root ganglion neurons

Necdin is expressed predominantly in postmitotic neurons and serves as a growth suppressor that is functionally similar to the retinoblastoma tumor suppressor protein. Using primary cultures of dorsal root ganglion (DRG) of mouse embryos, we investigated the involvement of necdin in the terminal differentiation of neurons. DRG cells were prepared from mouse embryos at 12.5 days of gestation and cultured in the presence of nerve growth factor (NGF). Immunocytochemistry revealed that necdin accumulated in the nucleus of differentiated neurons that showed neurite extension and expressed the neuronal markers microtubule-associated protein 2 and synaptophysin. Suppression of necdin expression in DRG cultures treated with antisense oligonucleotides led to a marked reduction in the number of terminally differentiated neurons. The antisense oligonucleotide-treated cells did not attempt to reenter the cell cycle, but underwent death with characteristics of apoptosis such as caspase-3 activation, nuclear condensation, and chromosomal DNA fragmentation. Furthermore, a caspase-3 inhibitor rescued antisense oligonucleotide-treated cells from apoptosis and significantly increased the population of terminally differentiated neurons. These results suggest that necdin mediates the terminal differentiation and survival of NGF-dependent DRG neurons and that necdin-deficient nascent neurons are destined to caspase-3-dependent apoptosis.