Search

β-Amyloid (Aβ) plaques can trigger chronic inflammation in the cellular environment that recruits infiltrating macrophages during the course of Alzheimer disease (AD). Activated macrophages release pro-inflammatory cytokines that increase neurotoxicity associated with AD. A major impediment to investigating neuroinflammation involving macrophage activity is the inability to discriminate resident microglial macrophages (mMϕ) from hematogenous macrophages (hMϕ), as they are morphologically and phenotypically similar when activated. To distinguish between mMϕ and hMϕ and to determine their respective roles in chronic inflammation associated with the progression of amyloidosis, we used lys-EGFP-ki transgenic mice that express enhanced green fluorescent protein in hMϕ, but not in mMϕ. These mice were crossed with 5XFAD mice. The offspring demonstrated robust AD pathology and enabled visual discrimination of mMϕ from hMϕ. Mutant mice demonstrated robust increases in Aβ1-42, area of Aβ plaques, gliosis and deficits in spatial learning by age 5 months. The time-course of Aβ accumulation, paralleled by the accumulation of hMϕ around Aβ plaques, was more robust in female compared with male mice and preceded behavioral changes. Thus, the accumulation of infiltrating hMϕ around Aβ plaques was age- and sex-dependent and preceded cognitive impairment., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

Cholinergic neurons rely on the sodium-dependent choline transporter CHT to provide choline for synthesis of acetylcholine. CHT cycles between cell surface and subcellular organelles, but little is known about regulation of this trafficking. We hypothesized that activation of protein kinase C with phorbol ester modulates choline uptake by altering the rate of CHT internalization from or delivery to the plasma membrane. Using SH-SY5Y cells that stably express rat CHT, we found that exposure of cells to phorbol ester for 2 or 5 min significantly increased choline uptake, whereas longer treatment had no effect. Kinetic analysis revealed that 5 min phorbol ester treatment significantly enhanced V(max) of choline uptake, but had no effect on K(m) for solute binding. Cell-surface biotinylation assays showed that plasma membrane levels of CHT protein were enhanced following 5 min phorbol ester treatment; this was blocked by protein kinase C inhibitor bisindolylmaleimide-I. Moreover, CHT internalization was decreased and delivery of CHT to plasma membrane was increased by phorbol ester. Our results suggest that treatment of neural cells with the protein kinase C activator phorbol ester rapidly and transiently increases cell surface CHT levels and this corresponds with enhanced choline uptake activity which may play an important role in replenishing acetylcholine stores following its release by depolarization., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Choline uptake by cholinergic nerve terminals is increased by depolarization; the literature suggests that this results from either the appearance of occult transporters or the increased activity of existing ones. The present experiments attempt to clarify the mechanism by which choline transport is regulated by testing if the preexposure of synaptosomes to choline mustard aziridinium ion prevents the stimulation-induced appearance of hemicholinium-3 binding sites and/or choline transport activity. Choline mustard inhibited irreversibly most of the "ground-state" (basal) high-affinity choline transport but only 50% of "ground-state" hemicholinium-3 binding sites. Exposure of both striatal and hippocampal synaptosomes to the mustard, before stimulation, inhibited K(+)-stimulated increases in choline transport and of [3H]-hemicholinium-3 binding. We conclude that the mechanism by which choline transport is regulated involves the increased activity of a pool of transport sites that are occluded to hemicholinium-3 but are available to choline mustard aziridinium ion, and presumably to choline, before stimulation. However, the concentration of mustard needed to inhibit the stimulation-induced increase of [3H]-hemicholinium-3 binding and choline transport was lower for striatal synaptosomes than for hippocampal synaptosomes. In the absence of extracellular Ca2+ or presence of high Mg2+ levels, the choline mustard did not prevent the appearance of extra striatal hemicholinium-3 binding sites. Also, high Mg2+ levels removed the ability of the mustard to inhibit K(+)-stimulated increases of either [3H]-hemicholinium-3 binding or choline transport by hippocampal synaptosomes. In contrast, the preexposure of hippocampal synaptosomes to the mustard in the presence of a calcium ionophore (A23187) reduced the concentration of inhibitor needed to prevent the activation of [3H]hemicholinium-3 binding and choline uptake. Thus, we conclude that the ability of the choline mustard to alkylate the pool of choline transporters that are activated by stimulation appears dependent on the entry of extra-cellular Ca2+.

NGF promotes the survival and enhances the neurotransmitter phenotype of basal forebrain and striatal cholinergic neurons in brain of rat. The objective of the present study was to determine whether stimulations of the cholinergic neuronal markers ChAT and high-affinity choline uptake are reflected in enhanced synthesis and release of ACh. Enhancement of ACh release in brain of adult and aged rats could result in increased cholinergic neurotransmission, and altered animal behavior. NGF (1.2 micrograms/d) was administered intracerebroventricularly for 2 weeks by osmotic minipump to male Fischer-344 rats aged 4 and 24 months. Cholinergic neuronal functional parameters were measured in frontal cortex, hippocampus, and striatum. In young adult rats, increased ChAT and choline uptake activities were accompanied by enhanced ACh synthesis, basal and depolarization-induced release of both endogenous and newly synthesized transmitter, with the largest effect generally being observed in striatum. In aged animals, the responses to NGF were less uniform. Whereas the pattern for changes in ChAT activity was similar to that seen in younger animals, choline uptake activity was increased only in frontal cortex and striatum. Coincidentally, ACh synthesis was also increased only in these two brain regions. ACh content of synaptosomes was not affected by age or NGF treatment, and the ACh levels in microwaved samples of striatum and basal forebrain were not affected by NGF treatment. However, profound deficits in both basal and evoked release of newly synthesized ACh were observed in the aged rats. NGF treatment had no significant effect on the basal release of newly synthesized ACh in aged rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to determine the effect of age and chronic intracerebral administration of nerve growth factor (NGF) on the activity of the presynaptic cholinergic neuronal markers hemicholinium-sensitive high-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) in the brain of Fisher 344 male rats. In 24-month-old rats, a substantial decrease in ChAT activity (30%) was measured in striatum, and decreases in HACU were found in frontal cortex (28%) and hippocampus (23%) compared with 4-month-old controls. Cholinergic neurons in brain of both young adult and aged rats responded to administration of exogenous NGF by increased expression of both phenotypes. In 4-month-old animals, NGF treatment at 1.2 micron/day resulted in increased activities of both ChAT and HACU in striatum (175 and 170%, respectively), frontal cortex (133 and 125%), and hippocampus (137 and 125%) compared with untreated and vehicle-treated 4-month-old animals; vehicle treatment had no effect on the activity of either marker. In 24-month-old animals treated with NGF for 2 weeks, ChAT activity was increased in striatum (179%), frontal cortex (134%), and hippocampus (119%) compared with 24-month-old control animals. Synaptosomal HACU in 24-month-old rats was increased in striatum (151%) and frontal cortex (128%) after 2 weeks of NGF treatment, but hippocampal HACU was not significantly different from control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrogen mustard analogues of choline are potent irreversible inhibitors of high-affinity choline transport at the cholinergic presynaptic nerve terminal in vitro. Ethylcholine mustard aziridinium ion, and to a lesser extent choline mustard aziridinium ion, have been used as tools to chemically lesion cholinergic neurons in the central nervous system. The selectivity of these compounds as neurotoxins for cholinergic neurons in vivo has been questioned and the mechanism by which they mediate neuronal death has not been elucidated. The objective of the present study was to investigate the selectivity of choline mustard aziridinium ion on embryonic rat brain neurons maintained in primary culture. The effect of choline mustard aziridinium ion on levels of cholinergic neuronal markers was compared with markers for GABAergic neurons as a measure of neuronal specificity. Choline mustard aziridinium ion at 10 and 30 microM irreversibly inhibited hemicholinium-sensitive, high-affinity choline transport into the cultured neurons without altering sodium-dependent, high-affinity GABA transport. Similarly, incubation of the neurons for 30 min in the presence of 10 microM choline mustard aziridinium ion led to a decrease in choline acetyltransferase activity of the cultures which was maintained for 72 h; glutamic acid decarboxylase activity was not altered under the same experimental conditions. Protein and DNA content and DNA-to-protein ratios of the choline mustard aziridinium ion-treated cultures were monitored as indicators of generalized cellular damage.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to measure the uptake and metabolism of [3H]choline mustard aziridinium ion in rat brain synaptosomes. In previous investigations, we showed that this compound binds irreversibly to the choline carrier thereby inhibiting choline transport into nerve terminals; it also acts as both a substrate and inhibitor of the acetylcholine biosynthetic enzyme choline acetyltransferase. We now report that [3H]choline mustard aziridinium ion was transported into purified rat brain synaptosomes by a hemicholinium-sensitive mechanism, but at only a fraction of the rate of uptake of [3H]choline. Following 5 min incubation with the nerve terminal preparation, uptake of [3H]choline mustard aziridinium ion was 20% of that of [3H]choline transport, but this fell to 10% of [3H]choline accumulation at 30 min incubation. Apparent Michaelis constants derived from double reciprocal plots of velocity of transport versus substrate concentration revealed that the apparent affinity constants (Km) of the high-affinity choline carrier for [3H]choline mustard aziridinium ion and [3H]choline were not different (1.44 +/- 0.15 and 2.14 +/- 0.80 microM for choline and choline mustard aziridinium ion, respectively). Increasing the incubation time from 5 to 30 min, during which time a proportion of the high-affinity choline carriers were irreversibly inactivated by choline mustard aziridinium ion, did not alter the binding affinity for this compound. The maximum velocity of transport (Vmax) for the two compounds were significantly different with the maximum uptake of [3H]choline mustard aziridinium ion being 19.5% of that for choline at 5 min incubation, and falling to only 10.6% of the maximum rate of choline transport by 30 min incubation. [3H]Choline mustard aziridinium ion transported into synaptosomes on the high-affinity choline carrier was metabolized, with 27% being recovered as [3H]acetylcholine mustard aziridinium ion, 27% as [3H]phosphorylcholine mustard aziridinium ion, 7% as unmetabolized [3H]choline mustard aziridinium ion and 16% recovered as an unidentified metabolite. In parallel samples, [3H]choline taken up into synaptosomes was recovered as [3H]acetylcholine (71%) and unmetabolized [3H]choline (18%) with no net production of [3H]phosphorylcholine. Acetylation of [3H]choline mustard aziridinium ion amounted to only 7.6% of [3H]acetylcholine synthesized under the same conditions. These results show clearly that choline mustard aziridinium ion was accumulated into the cholinergic nerve terminals by the high-affinity choline carrier, but the amount was small relative to the uptake of choline and probably restricted by progressive inactivation of the transporters through covalent bond formation.

Deficiencies in cholinergic nerve function have been clearly documented in patients with Alzheimer's disease. The lack of this neurotransmitter may be responsible for early memory loss in patients with the disease. Choline mustard Az ion has been used in our laboratory to produce cholinergic hypofunction in rat brain. Injections of the compound into the medial septal nucleus and dorsal hippocampus produced tissue lesions. The lesions were dose and time-dependent. Lesions produced in the medial septum resulted in transmitter depression in the hippocampus. These results suggest non-specific tissue damage because 5-HT levels were lower than normal. Other strategies for getting choline mustard Az ion into rat brain are being investigated to circumvent the apparent ability of this compound and other nitrogen mustard analogs of choline to produce non-specific tissue damage when injected directly into brain tissue.

Choline mustard aziridinium is a potent, irreversible and selective blocker of sodium-dependent, high-affinity transport of choline into rat forebrain synaptosomes; it was found to be 30 times less potent against low-affinity transport of choline. The IC50 value for high-affinity transport was 0.94 microM, compared to 29 microM for low-affinity uptake. The inhibitory action of choline mustard aziridinium ion on high-affinity transport of choline was graded with respect to time; a 12-fold increase in potency was obtained by increasing the inhibitor preincubation times from 1 to 30 min. Low concentrations of choline mustard aziridinium ion could produce significant blockade of choline carriers providing the exposure time was prolonged. The characteristics of the blockade of synaptosomal high-affinity choline transport by choline mustard aziridinium ion also changed depending upon preincubation time. The kinetics of inhibition of high-affinity choline transport by choline mustard aziridinium ion showed apparent competitive inhibition initially, followed by noncompetitive characteristics at longer preincubations with inhibitor. The rate of irreversible inhibition of carriers by this nitrogen mustard analogue would appear to be rapid; the rate constant was determined to be 5 X 10(-2) s-1 for micromolar concentrations of inhibitor. This action may preclude the transport of the mustard analogue into the nerve terminal, although initially some reversible binding with the carrier may result in the translocation of some choline mustard aziridinium ion into the presynaptic ending. The progressive alkylation of high-affinity carriers by the analogue could indicate the presence of excess carrier sites in the presynaptic membrane, or subpopulations of carriers in an inactive state in equilibrium with active carriers. A model is described for the inhibitory action of choline mustard aziridinium ion on synaptosomal high-affinity choline carriers.

The objectives of the present study were to validate the presence of cytoplasmic and membrane-associated pools of choline acetyltransferase (ChAT) in rat brain synaptosomes, and to evaluate inhibition of these different forms of the enzyme by the nitrogen mustard analogue of choline, choline mustard aziridinium ion (ChM Az). The relative distribution of ChAT and lactate dehydrogenase (LDH) was followed in subfractions of synaptosomes to establish whether ChAT activity associated with salt-washed presynaptic membranes represents membrane-bound protein rather than cytosolic enzyme trapped within undisrupted synaptosomes or revesiculated membrane fragments. The percentage of total synaptosomal ChAT activity (14%) recovered in the final membrane pellet always exceeded that of LDH (6%), lending support to the hypothesis that much of the ChAT associated with the membranes was a membrane bound form of the enzyme. Incubation of purified synaptosomes with ChM Az led to irreversible inhibition of ChAT activity; this loss of enzyme activity could not be accounted for by lysis of nerve terminals during incubation in the presence of the mustard analogue. Subfractionation of the ChM Az-treated nerve terminals revealed that the membrane-bound form of ChAT was inhibited to the greatest extent, followed by the ionically membrane-associated enzyme, with the activity of the water-solubilized enzyme not differing significantly from control. Preparation of the synaptosomal ChAT subfractions from untreated nerve terminals prior to incubation with varying concentrations of ChM Az or naphthylvinylpyridine revealed that under these conditions water-solubilized, ionically membrane-associated, and detergent-solubilized membrane-bound pools of ChAT were not differentially inhibited by either compound.(ABSTRACT TRUNCATED AT 250 WORDS)