Peroxynitrite donor SIN-1 alters high-affinity choline transporter activity by modifying its intracellular trafficking.

Sodium-coupled, high-affinity choline transporters (CHTs) are inhibited by 3-morpholinosydnonimine (SIN-1) [peroxynitrite (ONOO⁻) donor]; ONOO⁻ can be produced from nitric oxide and reactive oxygen species during neurodegeneration. SIN-1 rapidly increases CHT internalization from the cell surface, and this correlates with decreased choline uptake. This study addresses mechanisms by which SIN-1 inhibits CHT function in human neuronal SH-SY5Y cells. Thus, mutant L531A-CHT, which does not constitutively internalize into cells by a clathrin-mediated process, is resistant to SIN-1 effects. This suggests that CHT inhibition is not due to oxidative-nitrosative inactivation of the protein and that decreased levels of cell surface CHT in SIN-1-treated cells is related to alterations in its trafficking and subcellular disposition. Dominant-negative proteins AP180C and dynamin-K44A, which interfere with clathrin-mediated and dynamin-dependent endocytosis, respectively, attenuate CHT inhibition by SIN-1. CHT in both vehicle- and SIN-1-treated cells colocalizes with Rab7, Rab9, and Lamp-1 in late endosomes and lysosomes to a similar extent. Lysosome inhibitors increase choline uptake, suggesting that CHT proteins are normally degraded by lysosomes, and this is not altered by oxidative stress. Unexpectedly, inhibitors of proteasomes, but not lysosomes, attenuate SIN-1-mediated inhibition of choline uptake, indicating that proteasomal degradation plays a role in regulating CHT disposition in SIN-1-treated cells. SIN-1 treatment also enhances CHT ubiquitination. Thus, CHT inhibition in SIN-1-treated cells is mediated by proteasomal degradation, which differs from inhibitory mechanisms for some neurotransmitter transporters under similar conditions. Increased oxidative-nitrosative stress in the microenvironment of cholinergic nerve terminals would diminish cholinergic transmission by reducing choline availability for ACh synthesis.

Substrate-induced internalization of the high-affinity choline transporter.

Cholinergic neurons are endowed with a high-affinity choline uptake system for efficient synthesis of acetylcholine at the presynaptic terminals. The high-affinity choline transporter CHT1 is responsible for choline uptake, the rate-limiting step in acetylcholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylcholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylcholine synthesis at the cholinergic presynaptic terminals.

Separate or sequential exposure to nicotine prenatally and in adulthood: persistent effects on acetylcholine systems in rat brain regions.

Nicotine is a developmental neurotoxicant but the proposed "sensitization-homeostasis" model postulates that even in adulthood nicotine permanently reprograms synaptic function. We administered nicotine to rats throughout gestation or in adulthood (postnatal days PN90-107), simulating plasma levels in smokers, with evaluations on PN105, PN110, PN120, PN130 and PN180. We assessed nicotinic acetylcholine receptor (nAChR) binding, choline acetyltransferase activity, a marker for acetylcholine (ACh) terminals, and hemicholinium-3 (HC3) binding to the choline transporter, an index of ACh presynaptic activity. Prenatal nicotine exposure elicited persistent deficits in HC3 binding in male cerebral cortex and female striatum, but little change in other parameters. Nicotine given in adulthood produced profound nAChR upregulation lasting 2 weeks after discontinuing treatment. Decrements in cerebrocortical and striatal HC3 binding emerged during withdrawal and persisted through PN180, indicative of reduced ACh synaptic activity. Prenatal nicotine did not evoke any major alterations in the response to nicotine given in adulthood. The effects seen here are substantially different from those found previously for nicotine given to adolescent rats, which showed more prolonged nAChR upregulation and profound, widespread and persistent deficits in markers of ACh synaptic function; for adolescents, prenatal nicotine exposure desensitized nAChR responses, exacerbated withdrawal-induced ACh functional deficits, and worsened the long-term outcome. Our results indicate that the effects of nicotine during prenatal or adolescent stages are indeed distinct from the effects in adults, but that even adults show persistent changes after nicotine exposure, commensurate with the sensitization-homeostasis model. These effects may contribute to lifelong vulnerability to readdiction.

Synthesis and biodistribution of new radiolabeled high-affinity choline transporter inhibitors [11C]hemicholinium-3 and [18F]hemicholinium-3.

The high-affinity choline transporter (CHT1) system is an attractive target for the development of positron emission tomography (PET) biomarkers to probe brain, cardiac, and cancer diseases. An efficient and convenient synthesis of new radiolabeled CHT1 inhibitors [(11)C]hemicholinium-3 and [(18)F]hemicholinium-3 by solid-phase extraction (SPE) technique using a cation-exchange CM Sep-Pak cartridge has been well developed. The preliminary evaluation of both tracers through biodistribution studies in 9L-glioma rats has been performed, and the uptakes in the heart and tumor were observed, while very low brain uptake was seen.

Evaluation of [11C]hemicholinium-15 and [18F]hemicholinium-15 as new potential PET tracers for the high-affinity choline uptake system in the heart.

[(11)C]Hemicholinium-15 ([(11)C]HC-15) and [(18)F]hemicholinium-15 ([(18)F]HC-15) have been synthesized as new potential PET tracers for the heart high-affinity choline uptake (HACU) system. [(11)C]HC-15 was prepared by N-[(11)C]methylation of the appropriate precursor, 4-methyl-2-phenyl-morpholin-2-ol, using [(11)C]CH(3)OTf in 55-70% radiochemical yield decay corrected to end of bombardment (EOB) and 2-3Ci/mumol specific activity at end of synthesis (EOS). [(18)F]HC-15 was prepared by N-[(18)F]fluoromethylation of the precursor using [(18)F]FCH(2)OTf in 20-30% radiochemical yield decay corrected to EOB and >1.0Ci/mumol specific activity at EOS. The biodistribution of both compounds was determined in rats at 20min post-intravenous injection, and the results show the heart region uptakes 1.32+/-0.75%ID/g in R-ventricle for [(11)C]HC-15 and 1.28+/-0.81%ID/g in L-ventricle for [(18)F]HC-15, respectively. The dynamic PET imaging studies of [(11)C]HC-15 in rats were acquired 60min post-intravenous injection of the tracer using the IndyPET-II scanner. For the blocking experiments, the rats were intravenously pretreated with 3.0mg/kg of unlabeled HC-15 prior to [(11)C]HC-15 injection. [(11)C]HC-15 rat heart PET studies show rapid heart uptake to give clear heart images. The rat heart PET blocking studies found no significant blocking effect. The dynamic PET studies in normal and ablated dogs were performed using Siemens PET scanner with [(13)N]NH(3), [(11)C]HC-15, and [(18)F]HC-15. PET studies in dogs of both [(11)C]HC-15 and [(18)F]HC-15 also show significant heart uptake and give images of the heart. However, there is no significant change in [(11)C]HC-15 L-ventricle uptake following radiofrequency ablation in the dog. These results suggest that the localization of HC-15 tracers in the heart is mediated by non-specific processes, and the visualization of HC-15 tracers on the heart is related to non-specific binding of HACU.

Choline transporter as a novel target for molecular imaging of cancer.

Abnormalities of choline processing in cancer cells have been used as a basis for imaging of cancer with positron emission tomography and magnetic resonance spectroscopy. In this study, the transport mechanism for choline was investigated in cultured PC-3 prostate cancer cells. Furthermore, tritiated hemicholinium 3 (HC-3), a well-known inhibitor of choline transport, was studied as a prototypic molecular imaging probe in PC-3 cells and 9L glioma-bearing rats. [(3)H]Choline uptake by PC-3 cells was found to have both facilitative and nonfacilitative components. Facilitative transport was characterized by partial sodium dependence and intermediate affinity (K(M) = 9.7 +/- 0.8 microM). HC-3 inhibited choline with a K(I) of 10.5+/- 2.2 microM. Ouabain (1 mM) caused a 94% reduction in choline uptake. At physiologic choline concentration, phosphocholine was the rapid and predominant metabolic fate. The binding of [(3)H]HC-3 to PC-3 cells was rapid and specific (competitively blocked with unlabeled HC-3). Biodistribution of [(3)H]HC-3 in 9L glioma-bearing rats showed the ranking of uptake to be kidney > lung > tumor > liver > skeletal muscle congruent with blood > brain. In comparison with [(14)C]choline, [(3)H]HC-3 showed over twofold higher tumor uptake and favorable uptake ratios of tumor to blood, tumor to muscle, tumor to lung, and tumor to liver. The data demonstrate the quantitative importance of an intermediate-affinity, partially sodium-dependent choline transport system on choline processing in PC-3 cancer cells. The biodistribution properties of [(3)H]HC-3 in tumor-bearing rats encourage the development of molecular imaging probes based on choline transporter binding ligands.

Gestational dexamethasone treatment elicits sex-dependent alterations in locomotor activity, reward-based memory and hippocampal cholinergic function in adolescent and adult rats.

Glucocorticoids are the consensus treatment for preventing respiratory distress syndrome in preterm infants but there is emerging evidence of subsequent neurobehavioral abnormalities, independent of somatic growth effects. Pregnant rats were given 0.2 mg/kg of dexamethasone, a dose commensurate with clinical use, on gestational days 17-19 and behavioral evaluations were made on the offspring in adolescence and adulthood. The dexamethasone groups had the same body weights as the controls but nevertheless displayed long-term, sex-selective alterations in locomotor and cognitive behaviors. In the figure-8 activity apparatus, dexamethasone treatment ablated the normal sex differences in locomotor activity by reducing values in females to the lower level typical of males; habituation of activity similarly was impaired in females, reducing the profile to match that of control males, while male rats in the dexamethasone group showed a partially feminized pattern of habituation. In the 8-arm radial maze, control rats displayed typical sex differences, with male rats performing more accurately than females. Dexamethasone treatment eliminated this normal dichotomy, delaying learning in males while improving performance in females to the level normally seen in control males. Finally, we assessed hippocampal [3H]hemicholinium-3 binding as a biomarker for cholinergic synaptic activity, and again found loss of sex differences in the dexamethasone group: values in males were increased to the higher levels typical of females. These results indicate that gestational treatment with dexamethasone obtunds the normal sex differences in neurochemistry and behavior that are typically seen in adolescence in adulthood, thus producing sex-selective alterations in activity, learning, and memory.

Altered localization of choline transporter sites in the mouse hippocampus after prenatal heroin exposure.

Prenatal heroin exposure disrupts hippocampal cholinergic synaptic function and related behaviors. Biochemical studies indicate an increase in the number of presynaptic high-affinity choline transporter (HACT) sites, as assessed by [3H]hemicholinium-3 (HC-3) binding. The present study was designed to assess whether this effect involves global upregulation of the transporter, or whether disruption occurs with a specific tempero-spatial distribution. Pregnant mice were given 10mg/kg per day of heroin subcutaneously on gestational days (GD) 9-18. Autoradiographic distribution of HC-3 binding sites was evaluated in the hippocampus of the offspring at postnatal days 15, 25, and 53. These results, suggestive of hippocampal "miswiring," are likely to explain the net impairment of cholinergic synaptic function after prenatal heroin exposure, despite the simultaneous upregulation of both presynaptic cholinergic activity and postsynaptic receptors. Understanding the subregional selectivity of hippocampal defects can lead to the development of strategies that may potentially enable therapeutic interventions to offset or reverse the neurobehavioral defects.

Chlorpyrifos exposure during neurulation: cholinergic synaptic dysfunction and cellular alterations in brain regions at adolescence and adulthood.

The developmental neurotoxicity of chlorpyrifos (CPF) involves multiple mechanisms, thus rendering the immature brain susceptible to adverse effects over a wide window of vulnerability. Earlier work indicated that CPF exposure at the neural tube stage elicits apoptosis and disrupts mitotic patterns in the brain primordium but that rapid recovery ensues before birth. In the current study, we assessed whether defects in cholinergic synaptic activity emerge later in development. CPF was given to pregnant rats on gestational days 9-12, using regimens devoid of overt maternal or fetal toxicity. We then examined subsequent development of acetylcholine systems and compared the effects to those on general biomarkers of cell development. Choline acetyltransferase (ChAT), a constitutive marker for cholinergic nerve terminals, was increased in the hippocampus and striatum in adolescence and adulthood. In contrast, hemicholinium-3 (HC-3) binding to the presynaptic choline transporter, an index of nerve impulse activity, was markedly subnormal. Furthermore, m2-muscarinic cholinergic receptor binding was significantly reduced, instead of showing the expected compensatory upregulation for reduced neural input. CPF also elicited delayed-onset alterations in biomarkers of cell packing density, cell number, cell size and neuritic projections, involving brain regions both with and without reductions in indices of cholinergic activity. In combination with earlier results, the current findings indicate that the developing brain, and especially the hippocampus, is adversely affected by CPF regardless of whether exposure occurs early or late in brain development, and that defects emerge in adolescence or adulthood even in situations where normative values are initially restored in the immediate post-exposure period.

Progressive brain dysfunction following intracerebroventricular infusion of beta(1-42)-amyloid peptide.

The behavioral, neurochemical and histological changes of rats subjected to 3 days treatment with intracerebroventricular infusion of beta-amyloid peptides(Abeta)(1-42) were investigated 20 days and 80 days after the surgery. Abeta(1-42) produced a dose-dependent and a time-dependent impairment in the spontaneous alternation performance in the Y-maze (spatial working memory), place navigation task in a water maze (spatial reference memory) and passive avoidance retention (non-spatial long-term memory) at doses of 10 and 20 microg/rat. The learning impairments were more severe at 80 days than 20 days after infusion of Abeta(1-42). At 25 days after the infusion, a significant decrease in hemicholinium-3 (HC-3) binding was observed only in the hippocampus, although choline acetyltransferase (ChAT) activity was unchanged in the brain regions tested as compared with the vehicle (Abeta(40-1)) treatment. In contrast, the reduction in ChAT activity 85 days after Abeta(1-42) infusion was significant in hippocampus and striatum. HC-3 binding was also significantly decreased in the posterior cortex, hippocampus and striatum. In the histological analysis, brain atrophy was observed inasmuch as ventricular enlargement and neuronal damage in the CA1 area of the hippocampus were seen 85 days after Abeta(1-42) infusion. These results suggest that the rats subjected to intracerebroventricular infusion of Abeta(1-42) suffered from progressive brain dysfunction, and could be useful as an animal model for evaluating the developmental processes at the early and/or middle stage of Alzheimer's-type dementia.

Persistent cholinergic presynaptic deficits after neonatal chlorpyrifos exposure.

The commonly-used organophosphate insecticide, chlorpyrifos (CPF), impairs brain cell development, axonogenesis and synaptogenesis. In the current study, we administered CPF to neonatal rats on postnatal (PN) days 1-4 (1 mg/kg) or PN11-14 (5 mg/kg), treatments that were devoid of overt toxicity. We then examined two cholinergic synaptic markers, choline acetyltransferase activity (ChAT) and [3H]hemicholinium-3 binding (HC-3) in the hippocampus, midbrain, striatum, brainstem and cerebral cortex in the juvenile (PN30) and young adult (PN60). Across all brain regions, CPF exposure evoked significant reductions in both markers, with larger effects on HC-3 binding, which is responsive to neuronal impulse activity, than on ChAT, a constitutive marker. Superimposed on the deficits, there were gender-selective effects and distinct regional disparities in the critical exposure period for vulnerability. In the hippocampus, either the early or late treatment regimen evoked decreases in ChAT but the early regimen elicited a much larger decrease in HC-3; effects persisted into adulthood. In the midbrain, CPF administration on PN1-4 elicited deficits similar to those seen in the hippocampus; however, exposure on PN11-14 elicited changes preferentially in females. Gender selectivity was also apparent in the striatum, in this case reflecting deficits in females after CPF treatment on PN1-4. In contrast, the effects of CPF on the brainstem were relatively more robust in males; effects in the cerebral cortex were less notable than in other regions. These results indicate that neonatal CPF exposure produces widespread deficiencies in cholinergic synaptic function that persist into adulthood. The effects are likely to contribute to gender-selective alterations in behavioral performance that persist or emerge long after the termination of exposure and well after the restoration of cholinesterase activity.

6-hydroxydopamine-induced lesions of dopaminergic neurons alter the function of postsynaptic cholinergic neurons without changing cytoskeletal proteins.

The neuropathological hallmarks of many neurodegenerative diseases are intraneuronal inclusions containing cytoskeletal proteins such as neurofilaments in Lewy bodies in Parkinson's disease and tau in neurofibrillary tangles in Alzheimer's disease. Dysfunction in dopaminergic and cholinergic systems also exist in both Alzheimer's disease and Parkinson's disease. Because the primary pathology in Parkinson's disease is localized to the dopaminergic system, we set out to determine if perturbations in cholinergic systems are a consequence of dopaminergic neuron loss. Therefore, following intracerebral microinjections of 6-hydroxydopamine in rats, the activity of cholinergic neurons was measured by hemicholinium binding in cholinergic terminal fields and perturbations in cytoskeletal proteins were examined in dopaminoceptive neurons using immunocytochemistry. The 6-hydroxydopamine injections robustly reduced the number of monoaminergic cell bodies in the lateral midbrain and dramatically decreased dopamine and its major metabolites in dopaminergic projection sites. This treatment increased hemicholinium binding in the prefrontal cortex (200%) and amygdala (284%); however, despite previous reports to the contrary, there were no increases in immunoreactivity for phosphorylated neurofilaments, microtubule-associated protein (MAP) 2, tau or paired helical filament (PHF) tau. This lack of an increase in cytoskeletal proteins was observed following either injections of moderate doses of the toxin directly into the medial forebrain bundle or after high doses were administered intracerebroventricularly. These results suggest that removal of dopaminergic inputs to the forebrain results in hyperactivity of the cholinergic systems but is not sufficient to induce postsynaptic perturbations in cytoskeletal proteins which occur in neurodegenerative diseases.

Role of high-affinity choline uptake on extracellular choline and acetylcholine evoked by NMDA.

Previous studies have shown that NMDA evokes a calcium-dependent and region-specific increase in extracellular choline that is associated with a reduction of membrane phosphatidylcholine and precedes neuronal cell death. We investigated, using in vivo microdialysis, the contribution of high-affinity choline uptake on the increase in extracellular choline evoked by NMDA. Dialysis was performed in the presence of Neostigmine (0.5 microM), an acetylcholinesterase inhibitor, in prefrontal cortex or hippocampus of freely moving rats. Drugs were administered through the dialysis probe. In cholinergic denervation experiments, rats were subjected to sham or AMPA-induced lesion of cholinergic nuclei at least 2 weeks before microdialysis. Excitotoxic lesion of the medial septum / ventral diagonal band nuclei reduced hippocampal choline acetyltransferase activity by 74%, [(3)H]hemicholinium-3 binding by 32%, and completely abolished potassium-evoked acetylcholine release. Despite this reduction of presynaptic cholinergic function, perfusion of NMDA (300 microM) by retrodialysis produced an increase in hippocampal extracellular choline (249 +/- 22% of basal levels) that was similar to that observed in sham controls (301 +/- 35%). Inhibition of choline uptake with hemicholinium-3 in nonlesioned rats produced a sustained increase in dialysate choline (163 +/- 8%) and reduced acetylcholine to 33 +/- 2% of basal levels, consistent with a depletion of the acetylcholine pool due to precursor deficit. Simultaneous perfusion of hemicholinium-3 and NMDA produced a synergistic increase in dialysate choline (664 +/- 95% of basal levels), indicating that part of the choline released by NMDA is taken up. In contrast, NMDA antagonized the decrease of acetylcholine produced by hemicholinium-3. These results show that NMDA-evoked choline release is not mediated by inhibition of high-affinity choline uptake and indicate that choline released by NMDA can be used to sustain acetylcholine synthesis when there is a precursor deficit secondary to uptake inhibition.

The involvement of protein kinase C in the regulation of choline cotransport in Limulus.

The involvement of protein kinase C (PKC) in the regulation of [3H]choline cotransport was studied in Limulus brain hemi-slice preparations. The PKC activators, phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu), significantly decreased [3H]choline cotransport. Conversely, the PKC inhibitors, staurosporine (STAURO) and polymyxin B (PMB), each increased [3H]choline cotransport. These PKC inhibitors prevented the phorbol ester-induced reduction of transport. Both the PMA induced decrease and the STAURO induced increase in [3H]choline cotransport were paralleled by respective and comparable changes in [3H]hemicholinium-3 (HC-3) specific binding. Pre-exposure of brain hemi-slices to elevated potassium chloride (120 mM KCl) resulted in a doubling of [3H]choline cotransport and [3H]HC-3 binding. The enhancement of [3H]choline cotransport by STAURO and antecedent 120 mM KCl treatment were additive. PMA did not significantly alter elevated potassium stimulated transport. Moreover, arachidonyltrifluoromethyl ketone (AACOCF3) and quinacrine (QUIN), both phospholipase A2 (PLA2) inhibitors, markedly decreased enhanced [3H]choline transport and [3H]HC-3 binding induced by antecedent exposure to depolarizing concentrations of potassium. These results suggest that PKC and PLA2 are involved in the regulation of [3H]choline cotransport but at different regulatory sites.

Transport of choline in rat brain slices.

Enzyme-modified amperometric microsensors have been utilized in the investigation of acetylcholine and choline diffusion in solution and choline uptake and diffusion in rat brains. A small amount of the substance of interest was introduced by pressure injection and transport to the sensor was monitored. The apparent diffusion coefficients for acetylcholine and choline in agarose gel perfused with physiological solutions were determined to be 5.2 +/- 0.7 x 10(-6) cm2/s and 6.1 +/- 0.8 x 10(-6) cm2/s, respectively. Choline transport was monitored in two brain regions: the caudate and anterior hypothalamus. The transport time of choline in the caudate was concentration dependent, but was unaffected by the presence of a competitive, high-affinity uptake inhibitor, hemicholinium-3. The apparent diffusion coefficient (D) and uptake rate (k) for choline in the caudate and anterior hypothalamus were calculated using a model for point source diffusion coupled with first-order uptake kinetics. The effect of the sensors' response time on the measurements was removed by deconvolution. The D and k were 1.8 +/- 0.1 x 10(-6) cm2/s and 2.0 +/- 0.1 x 10(-2) s-1 in the caudate and 1.9 +/- 0.1 x 10(-6) cm2/s and 3.2 +/- 0.6 x 10(-2) s-1 in the anterior hypothalamus. The reduced diffusion coefficient determined in brain tissue compared to agar gel is consistent with the increased tortuosity of the brain microenvironment. A substance in brain tissue, presumably acetylcholinesterase, prevents the use of differential measurements of acetylcholine because choline sensors became sensitive to acetylcholine.

Cholinergic markers in degenerative parkinsonism: autoradiographic demonstration of high-affinity choline uptake carrier hyperactivity.

[3H]Hemicholinium-3 ([3H]HC-3) binding, as a marker of the presynaptic high-affinity choline uptake carrier (HACU), and cholinergic muscarinic receptors were measured by autoradiography in several brain regions of levodopa-responsive parkinsonism and matched cases. A significant increase in the density of [3H]HC-3 binding sites was found in the striatum of parkinsonian brains, while there was a slight decrease in the parkinsonian hippocampus. Total, M1 and non-M1 muscarinic receptors remained unchanged in frontal cortex and striatum of parkinsonian brains as compared to controls. Total and non-M1 muscarinic receptors were significantly reduced in the parkinsonian hippocampus, whereas hippocampal M1 receptors were preserved. These data demonstrate a hyperactivity of the HACU, and thus of the acetylcholine synthesis, in parkinsonian brains probably compensatory of the loss of both dopaminergic terminals in the striatum and of basal forebrain neurons in the hippocampus. Our results emphasize the value of [3H]HC-3 binding in the study of the functional status of the cholinergic synapse in neurodegenerative disorders.