Clonal Nerve Research Articles

Amyloid Beta Resistance in Nerve Cell Lines Is Mediated by the Warburg Effect

Amyloid beta (Aβ) peptide accumulation in the brains of patients with Alzheimer's disease (AD) is closely associated with increased nerve cell death. However, many cells survive and it is important to understand the mechanisms involved in this survival response. Recent studies have shown that an anti-apoptotic mechanism in cancer cells is mediated by aerobic glycolysis, also known as the Warburg effect. One of the major regulators of aerobic glycolysis is pyruvate dehydrogenase kinase (PDK), an enzyme which represses mitochondrial respiration and forces the cell to rely heavily on glycolysis, even in the presence of oxygen. Recent neuroimaging studies have shown that the spatial distribution of aerobic glycolysis in the brains of AD patients strongly correlates with Aβ deposition. Interestingly, clonal nerve cell lines selected for resistance to Aβ exhibit increased glycolysis as a result of activation of the transcription factor hypoxia inducible factor 1. Here we show that Aβ resistant nerve cell lines upregulate Warburg effect enzymes in a manner reminiscent of cancer cells. In particular, Aβ resistant nerve cell lines showed elevated PDK1 expression in addition to an increase in lactate dehydrogenase A (LDHA) activity and lactate production when compared to control cells. In addition, mitochondrial derived reactive oxygen species (ROS) were markedly diminished in resistant but not sensitive cells. Chemically or genetically inhibiting LDHA or PDK1 re-sensitized resistant cells to Aβ toxicity. These findings suggest that the Warburg effect may contribute to apoptotic-resistance mechanisms in the surviving neurons of the AD brain. Loss of the adaptive advantage afforded by aerobic glycolysis may exacerbate the pathophysiological processes associated with AD.

The Regulation of Glucose Metabolism by HIF-1 Mediates a Neuroprotective Response to Amyloid Beta Peptide

It is frequently argued that both amyloid beta (Aβ) and oxidative stress are involved in the pathogenesis of Alzheimer's disease (AD). We show here that clonal nerve cell lines and primary cortical neurons that are resistant to Aβ toxicity have an enhanced flux of glucose through both the glycolytic pathway and the hexose monophosphate shunt. AD brain also has increased enzymatic activities in both pathways relative to age-matched controls. The Aβ-induced changes in glucose metabolism are due to the activation of the transcription factor hypoxia inducible factor 1 (HIF-1). As a result of Aβ-induced changes in glucose metabolism, Aβ-resistant cells are more readily killed by glucose starvation and by classes of antipsychotic drugs that inhibit glucose uptake.

The Activation of Dopamine D4 Receptors Inhibits Oxidative Stress-Induced Nerve Cell Death

Oxidative stress is thought to be the cause of nerve cell death in many CNS pathologies, including ischemia, trauma, and neurodegenerative disease. Glutamate kills nerve cells that lack ionotropic glutamate receptors via the inhibition of the cystine-glutamate antiporter x(c)(-), resulting in the inhibition of cystine uptake, the loss of glutathione, and the initiation of an oxidative stress cell death pathway. A number of catecholamines were found to block this pathway. Specifically, dopamine and related ligands inhibit glutamate-induced cell death in both clonal nerve cell lines and rat cortical neurons. The protective effects of dopamine, apomorphine, and apocodeine, but not epinephrine and norepinephrine, are antagonized by dopamine D4 antagonists. A dopamine D4 agonist also protects, and this protective effect is inhibited by U101958, a dopamine D4 antagonist. Although the protective effects of some of the catecholamines are correlated with their antioxidant activities, there is no correlation between the protective and antioxidant activities of several other ligands. Normally, glutamate causes an increase in reactive oxygen species (ROS) and intracellular Ca(2+). Apomorphine partially inhibits glutamate-induced ROS production and blocks the opening of cGMP-operated Ca(2+) channels that lead to Ca(2+) elevation in the late part of the cell death pathway. These data suggest that the protective effects of apomorphine on oxidative stress-induced cell death are, at least in part, mediated by dopamine D4 receptors via the regulation of cGMP-operated Ca(2+) channels.

A Role for 12-lipoxygenase in Nerve Cell Death Caused by Glutathione Depletion

An early and highly specific decrease in glutathione (GSH) in the substantia nigra is associated with Parkinson's disease, and low levels of GSH lead to the degeneration of cultured dopaminergic neurons. Using immature cortical neurons and a clonal nerve cell line, it is shown that a decrease in GSH triggers the activation of neuronal 12-lipoxygenase (12-LOX), which leads to the production of peroxides, the influx of Ca2+, and ultimately to cell death. The supporting evidence includes: 1) inhibitors of arachidonate metabolism and 12-LOX block cell death induced by GSH depletion; 2) there is an increase in 12-LOX activity and a membrane translocation in HT22 cells, and an induction of the enzyme in primary cortical neurons following the reduction of GSH; 3) 12-LOX is directly inhibited by GSH; and 4) exogenous arachidonic acid potentiates cell death. These data show that the LOX pathway is a critical intermediate in at least some forms of neuronal degeneration.

The expression of amyloid beta protein precursor protects nerve cells from β-amyloid and glutamate toxicity and alters their interaction with the extracellular matrix

The biological function of amyloid β protein precursor (ABPP) in nerve cells is examined by transfecting it into the B103 clonal nerve cell line which makes no ABPP and asking if a variety of biological activities are altered. Although ABPP is expressed by the transfected cells at high levels, there are no detectable differences between the ABPP negative control clones and the clones expressing ABPP with respect to growth rate in high serum medium, dibutyryl-cyclic AMP-induced differentiation, or morphology on plastic culture dishes. There are, however, significant differences in a number of properties between the two groups of cell lines. Cells expressing ABPP adhere more rapidly and have an enhanced rate of neurite outgrowth on collagen substrata. They also grow more rapidly in low serum medium. Finally, the expression of ABPP weakly but significantly protects the nerve cells from amyloid β protein and glutamate toxicity.

Nerve growth factor-induced neuronal differentiation is accompanied by differential splicing of β-amyloid precursor mRNAs in the PC12 cell line

The effect of the neurotrophic factor nerve growth factor (NGF) on the expression of the β-amyloid gene has been studied in the clonal nerve cell line PC12. The neuronal differentiation of PC12 cells in the presence of NGF was accompanied by a shift in the ratio of β-amyloid precursor protein (APP) transcripts. In particular there was reduced expression of the transcript coding for the longest precursor form (APP 770) and a concomitant increase in the shortest (APP 695) transcript following NGF treatment.

3H-noradrenaline release potentiated in a clonal nerve cell line by low-intensity pulsed magnetic fields

Recent studies have shown that extremely low-frequency (ELF) electric fields1 and radiofrequency amplitude-modulated electromagnetic fields2,3,25 can influence ion fluxes in neural tissue. Furthermore, the application of low-frequency magnetic (LFM) fields in the successful treatment of non-union fractures and pseudo-arthrosis has been widely reported4–6. Stimulated by these findings and the wide variety of effects reported in other biological systems7–10, we have set out to establish a model system for studying the biological effects of such low-frequencyfields that readily allows analysis of their mechanism of action. Clonal lines in tissue culture are an obvious choice for such a model, first, because the analysis is simplified by the presence of only one cell type and second, because the geometry of the biological sample can be specified to allow precise quantitation of applied field strengths and induced current densities. One such clonal line, designated PC12, is a likely candidate for such an approach as it expresses many properties of differentiated sympathetic neurones11–16, including calcium-dependent release of newotransmitters11,12,14. We report here that 3H-noradrenaline(NA) release from PC12 cells is stimulated by an inductively coupled 500-Hz LFM field with a magnitude comparable with certain cholinergic stimuli in this system.

Regulation of acetylcholinesterase by nerve growth factor in the pheochromocytoma PC12 cell line

In the PC12 clonal nerve cell line derived from a rat pheochromocytoma, the activity of acetylcholinesterase (AChE) is regulated by nerve growth factor (NGF). The specific activity of AChE was significantly increased 2 days after exposure of PC12 cells to NGF (8 × 10 −8 M), and reached its maximal increase of 94% after 3 days. Thereafter the specific activity of AChE did not significantly vary during the following 7 days. AChE cytochemistry showed the enzyme activity to be localized predominantly in cisternae of the rough endoplasmic reticulum, with a strongly enhanced activity after NGF treatment.

Binding properties of a neurotoxin from the venom of the green mamba, Dendroaspis viridis.

A toxin, alpha-mambatoxin, was purified from the venom of the green mamba Dendroaspis viridis using the procedures of Shipolini et al. (Shipolini, R. A., Bailey, G. S., Edwardson, J. A., and Banks, E. C. (1973) Eur. J. Biochem. 40, 337-344). The purified toxin blocks agonist-induced activation of acetylcholine receptors on muscle cells but, like alpha-bungarotoxin, it does not affect agonist-induced activation of receptors on a clonal sympathetic nerve cell line (PC12), an endothelial cell line, cultured chick ciliary ganglion neurons, or frog cardiac ganglion neurons. The toxin does block binding of alpha-bungarotoxin to cultures of muscle and nerve, and iodinated alpha-mambatoxin binds to cultures of muscle and nerve. The alpha-mambatoxin binding component on muscle was identified as acetylcholine receptor on the basis of sedimentation, immunoprecipitation, and rate of degradation. The alpha-mambatoxin binding component on PC12 cells, like the alpha-bungarotoxin binding component on these cells, is not recognized by anti-acetylcholine receptor antisera which do recognize acetylcholine receptor on these cells. The number of alpha-mambatoxin binding sites on both nerve and muscle when assayed in situ is twice that of alpha-bungarotoxin binding sites. However, when muscle cells are solubilized in nonionic detergents and then labeled with toxins, the number of alpha-mambatoxin binding sites is reduced and the two toxins bind in equal molar amounts. Finally, unlike alpha-bungarotoxin, which dissociates from complexes formed with nerve, alpha-mambatoxin forms complexes with nerve which dissociate only very slowly if at all.

GABA and picrotoxinin receptors in clonal nerve cells.

γAminobutyric acid (GABA) is a neurotransmitter which seems to mediate inhibitory synaptic transmission in many regions of the central nervous system (CNS) of vertebrates and invertebrates1. GABA has been shown to act by increasing membrane chloride permeability in invertebrate muscle2,3, and a similar mechanism has been suggested for the vertebrate CNS4. The response to GABA is inhibited noncompetitively by the convulsant picrotoxinin1–5. Binding sites for radioactive GAB A6–8 and picrotoxinin9–11 (the active component of picrotoxin) have been described in homogenates of invertebrate muscle and mammalian CNS; these binding sites show many properties expected of postsynaptic membrane macromolecules involved in the receptor-ionophore function of GABA. Nevertheless, unambiguous correlation between biochemical assays and in vivo function is difficult to obtain, especially in heterogeneous tissue such as mammalian CNS. One approach to this problem involves cloned cell lines of neuronal origin which retain in culture the differentiated function to be studied (see, for example, ref. 12). Certain cell lines cloned from rat13 and mouse14 nervous system possess neuronal properties such as electrical excitability, neurotransmitter synthetic enzymes12, neurotransmitter uptake15,16, binding of defined ligands to their appropriate neurotransmitter receptors12,17,18, and receptor-coupled responses to neurotransmitters17,19,20. Several cell lines of apparent glial origin have also been described13,21,22. To determine whether binding sites for GABA and picrotoxinin are localised to nerve cells, a variety of clonal cell lines were assayed for their ability to bind those ligands. We report here the presence of GABA and picrotoxinin binding sites with receptor-like properties in several clonal nerve cell lines obtained from the rat CNS, and the absence of such binding sites in many other cell lines of non-neuronal origin.

Substance P enhances cholinergic receptor desensitization in a clonal nerve cell line.

Substance P inhibits carbamylcholine-induced 22Na+ uptake in the clonal cell line PC12. This inhibition is noncompetitive with agonist but competitive with Na+. Octahydrohistrionicotoxin (H8-HTX) also exhibits this same pattern of inhibition. Moreover, both substance P and H8-HTX are very effective in enhancing agonist-induced receptor desensitization. Local anesthetics, such as QX222, also cause inhibition that is competitive with Na+, but they have only marginal effects on desensitization. Because substance P and H8-HTX cannot by themselves cause desensitization, their action is dependent on and synergistic with the action of agonist. Furthermore, substance P and H8-HTX do not appear to compete for the same site as QX222, which is thought to bind to the ion channel. Finally, substance P can stabilize the desensitized state of the receptor even when added subsequent to the actual desensitization and removal of agonist. Thus, substance P does not require open ion channels for binding and may modulate the activity of the receptor-ionophore complex by binding to a distinct regulatory site.

Sodium and calcium fluxes in a clonal nerve cell line.

1. 22Na+ and 45Ca2+ fluxes were studied in the clonal nerve cell line PC12. Three distinct types of ion channels were found: (a) voltage-dependent Na+ channels, (b) voltage-dependent Ca2+ channels, and (c) acetylcholine-activated channels permeable to both ions. 2. 22Na+ uptake through voltage-dependent Na+ channels is induced by veratridine and scorpion venom, and is inhibited 50% by 5 X 10(-7) M-tetrodotoxin and greater than 98% by 5 X 10(-6) M-tetrodotoxin. 3. 45Ca2+ uptake through voltage-dependent Ca2+ channels is induced by depolarizing the cells in 50 mM-KCl. This flux is not dependent on the presence of Na+ in the medium and is insensitive to 5 X 10(-6) M-tetrodotoxin. However, 1 mM-Mn2+ causes a 95% inhibition of K+-induced 45Ca2+ uptake. 4. Veratridine and scorpion venom also induce voltage-dependent 45Ca2+ uptake which can be blocked by 1mM-Mn2+. In contrast to KCl-induced 45Ca2+ uptake, this flux is completely blocked by 5 X 10(-6) M-tetrodotoxin and is abolished by removal of Na+ from the medium. Thus the depolarizing stimulus for Ca2+ uptake in this case is Na+ from the medium. Thus the depolarizing stimulus for Ca2+ uptake in this case is Na+ influx through voltage-dependent Na+ channels. 5. Carbamylcholine induces both 22Na+ and 45Ca2+ fluxes which are blocked by nicotinic cholinergic antagonists with the exception of alpha-bungarotoxin. The 22Na+ flux occurs exclusively via acetylcholine receptor channels, as evidenced by the lack of effect of 5 X 10(-6) M-tetrodotoxin. In the presence of Na+, almost all of the 45Ca2+ uptake can be blocked by 1 mM-Mn2+ and thus occurs via voltage-dependent Ca2+ channels which are activated by the depolarizing Na+ influx. 6--8% of the total 45Ca2+ flux, however, is insensitive to 1 mM-Mn2+, suggesting that this portion of the uptake occurs via the acetylcholine receptor channels. In Na+-free medium, the Mn2+-resistant 45Ca2+ component increases to 40% of the total uptake, apparently due to lack of competition from Na+ for the acetylcholine receptor channels. This receptor-linked flux still causes sufficient depolarization to induce the additional 60% of the Ca2+ flux through voltage-dependent, Mn2+ sensitive Ca2+ channels. 6. Mn2+ inhibits Ca2+ flux through voltage-dependent Ca2+ channels by competing for entry through these channels. 50 mM-KCl induces 54Mn2+ fluxes in PC12 cells that are comparable in magnitude to 45Ca2+ fluxes. 7. In normal saline 45Ca2+ efflux from PC12 cells is several times more rapid than in Na+-free medium, indicating the presence of a Ca2+-Na+ exchange mechanism.

NGF-induced alternations in protein secretion and substrate-attached material of a clonal nerve cell line

NGF-induced alternations in protein secretion and substrate-attached material of a clonal nerve cell line

Neurotransmitter Regulation of Adenosine 3′,5′-Monophosphate in Clonal Nerve, Glia, and Muscle Cell Lines

Norepinephrine increases the intracellular level of adenosine 3',5'-monophosphate (cyclic AMP) in clonal cell lines of nerve, glia, smooth muscel, and skeletal muscle. The largest response is in skeletal muscle, where the cyclic nucleotide concentration is elevated more than 500-fold. Glia and muscle cells, but not nerve cells, respond to dopamine with increased cyclic AMP accumulation. This response appears to be mediated through a beta-adrenoreceptor.

Multiple modes of dibutyryl cyclic AMP-induced process formation by clonal nerve and glial cells

Exposure of clonal nerve and glial cells to dibutyryl adenosine-3′,5′-monophosphate (db-cAMP) induced processes. These cellular processes were generated either by the extension of a process (growth) or by retraction of parts of an extended cytoplasm (shrinkage). In all cases, these cells from the central nervous system rounded up in response to db-cAMP, rather than spread on the substratum.

The uptake of gaba by clonal nerve and glia

A number of clonal nerve and glial cell lines are able to accumulate exogenous GABA. The ability of the cells to concentrate GABA is dependent upon external calcium, while the uptake process is sodium dependent and temperature sensitive. GABA uptake by the cell lines is specific with respect to analogue inhibition, but this specificity is different from that observed in brain slices. It is, however, similar to that of glia in the rat sensory ganglia. Although clonal muscle cells can concentrate GABA to a limited extent, a number of other differentiated cells cannot.

Neurotransmitter synthesis by two clonal nerve cell lines: with culture growth and morphological differentiation

Neurotransmitter synthesis by two clonal nerve cell lines: with culture growth and morphological differentiation

Role of Muscle Activity in Nerve-Muscle Interaction in vitro

CLONAL nerve and muscle cells interact in vitro to produce a nerve-muscle contact which is associated with an area of increased acetylcholine sensitivity on the muscle1,2. This association is reminiscent of the distribution of acetylcholine sensitivity on innervated skeletal muscle in vivo3,4. It is possible to use cultures of nerve and muscle cells to examine the requirements for localization in vitro. A previous report showed that cholinergic transmission is not required2. Here I discuss the requirement for muscle electrical and contractile activity in localisation of acetylcholine sensitivity.