Cholinergic deficit in Alzheimer's disease: A study based on CSF and autopsy data

Cholinesterase (ChE) activity was measured as a possible marker of cholinergic neurotransmission of the brain in CSF of 93 patients with probable Alzheimer's disease/senile dementia of the Alzheimer type (AD/SDAT) and of 29 control patients. ChE activity in CSF was decreased significantly in the AD/SDAT patients as compared to the controls. This reduction correlated significantly with the various measures of the severity of dementia. However, the reduction of ChE activity was only moderate (25–30%) even in patients with the most severe dementia and nonsignificant in patients with early symptoms of AD/SDAT. The significance of various confounding factors, which may interfere with CSF ChE measurements is discussed. Our findings seem to indicate that the deficiency of cholinergic neurons is not directly reflected in CSF and that the measurements of ChE activities in CSF are not helpful in diagnosing AD/SDAT. In the autopsy study the activities of cholineacetyltransferase (ChAT) and ChE were determined for ten brain areas of 20 AD/SDAT patients and of 14 controls. In AD/SDAT patients ChAT activity was profoundly decreased (50–85% decrease) in the cortical areas and hippocampus, but was unchanged or only mildly reduced in other subcortical brain areas. This study further confirms that the affection of cholinergic neurons is limited to projections from nucleus basalis to cortex and hippocampus, whereas other cholinergic neurons, like in striatum, seem to be relatively spared. In general, the activities of ChAT and ChE were lower in Alzheimer patients dying at younger age suggesting more severe disease process with these patients.     

Regulated Extracellular Choline Acetyltransferase Activity— The Plausible Missing Link of the Distant Action of Acetylcholine in the Cholinergic Anti-Inflammatory Pathway

Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT) in human cerebrospinal fluid (CSF) and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer’s disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic signaling in states of inflammation in general and in neurodegenerative disease, such as Alzheimer’s disease and multiple sclerosis in particular.     

Phenotype dependent differential effects of interleukin-1beta and amyloid-beta on viability and cholinergic phenotype of T17 neuroblastoma cells

Amyloid-beta accumulation in brains of Alzheimer's disease (AD) victims is accompanied by glial inflammatory reactions and preferential loss of cholinergic neurons. Therefore, the aim of this study was to find out whether proinflamatory cytokine interleukin 1beta (IL1beta) modifies effects of amyloid-beta (Abeta) on viability and cholinergic phenotype of septum derived T17 cholinergic neuroblastoma cells. In nondifferentiated T17 cells (NC) Abeta(25-35) (1 microg/ml) caused no changes in choline acetyltransferase (ChAT) activity, acetylcholine (ACh) release, subcellular distribution of acetyl-CoA, but doubled content of trypan blue positive cells. IL1beta (10 ng/ml) increased ACh release (125%) but did not change other parameters of NC. In the presence of Abeta IL1beta also increased ChAT activity (47%), ACh release (100%) but had no effect on acetyl-CoA distribution and cell viability. Differentiation with retinoic acid and dibutyryl cyclic AMP caused over two-fold increase of ChAT activity and ACh content, four-fold increase of ACh release and about 50% decrease of acetyl-CoA level in the mitochondria. In differentiated cells (DC), Abeta decreased ChAT activity (31%), ACh release (47%) and content of acetyl-CoA (80%) in cell cytoplasmic compartment, whereas IL1beta elevated ChAT activity (54%) and ACh release (32%). IL1beta totally reversed Abeta-evoked inhibition of ChAT activity and ACh release and restored control level of cytoplasmic acetyl-CoA but increased fraction of nonviable cells to 25%. Thus, IL1beta could compensate Abeta-evoked cholinergic deficits through the restoration of adequate expression of ChAT and provision of acetyl-CoA to cytoplasmic compartment in cholinergic neurons that survive under such pathologic conditions. These data indicate that IL1beta possess independent cholinotrophic and cholinotoxic activities that may modify Abeta effects on cholinergic neurons.     

Properties of choline acetyltransferase from the bulb miteRhizoglyphus echinopus (Acari: Acaridae)

In the presence of exogenous acetyl coenzyme A¹⁴-C and choline, the 20 000 g supernatant fraction of whole bulb-mite homogenates synthesized a radioactive product that chromatographed with authentic acetylcholine (ACh). ApparentK _m values were 0.12 and 1.14 mM for acetyl coenzyme A and choline, respectively, and the average rate of ACh synthesis was 2.06 mol h^–1 g^–1 equivalent of mite tissue (wet weight). Choline acetyltransferase (ChAT) activity was inhibited by 5,5-dithiobis(2-nitrobenzoic acid), suggesting the presence of functionally important sulfhydryl groups. However, ChAT activity was not inhibited by 27 acaricides from several different chemical classes, except for the thiazolidine flubenzimine which gave a pI₅₀ of 3.5 mol l^–1.Contribution from the Missouri Agricultural Experiment Station, Columbia, Missouri, Journal Series No. 11 157.     

Roles played by lymphocyte function-associated antigen-1 in the regulation of lymphocytic cholinergic activity

Lymphocytes possess the essential components of a cholinergic system, including acetylcholine (ACh); choline acetyltransferase (ChAT), its synthesizing enzyme; and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Stimulation of lymphocytes with phytohemagglutinin, which activates T cells via the T cell receptor/CD3 complex, enhances the synthesis and release of ACh and up-regulates expression of ChAT and M(5) mAChR mRNAs. In addition, activation of protein kinase C and increases in intracellular cAMP also enhance cholinergic activity in T cells, and lymphocyte function associated antigen-1 (LFA-1; CD11a/CD18) is an important mediator of leukocyte migration and T cell activation. Anti-CD11a monoclonal antibody (mAb) as well as antithymocyte globulin containing antibodies against CD2, CD7 and CD11a all increase ChAT activity, ACh synthesis and release, and expression of ChAT and M(5) mAChR mRNAs in T cells. The cholesterol-lowering drug simvastatin inhibits LFA-1 signaling by binding to an allosteric site on CD11a (LFA-1 alpha chain), which leads to immunomodulation. We found that simvastatin abolishes anti-CD11a mAb-induced increases in lymphocytic cholinergic activity in a manner independent of its cholesterol-lowering activity. Collectively then, these results indicate that LFA-1 contributes to the regulation of lymphocytic cholinergic activity via CD11a-mediated pathways and suggest that simvastatin exerts its immunosuppressive effects in part via modification of lymphocytic cholinergic activity.     

Recovery of acetylcholine release and choline acetyltransferase activity after freezing-induced degeneration of rat soleus muscle

In order to study the effect of synaptic contact on the amounts of choline acetyltransferase (ChAT) and acetylcholine (ACh) in the nerve terminals and on their ability to release ACh, a freeze—thaw procedure was developed as a means to induce long lasting degeneration of rat soleus muscle. It was found that 4 days after the freeze—thaw procedure the preparation did not contract upon direct electric stimulation and the level of creatine kinase (CK) was below detection. The preparation contained about 15% of the ChAT activity and 15% of the ACh content of the controls. The ACh release evoked by 50 mM KCl was 25% of controls, but it was, when expressed as a fraction of the ACh content, about twice as high as that in control muscles. At day 12, the preparation still did not contract and the level of CK was less than 5% of controls. The ChAT activity and the ACh content were 40%) and 20% of controls, respectively. However, no release of ACh could be evoked by 50 mM KCl. At days 28 and 58 the preparation contracted upon stimulation of the nerve; the CK activity had recovered to about 20% and the ACh content to 40%, while the ChAT activity did not increase above 40%. The KCl–evoked ACh release had recovered to 20—30% of controls. The results indicate that freezing destroyed muscle cells and most intramuscular nerve branches. Subsequent regeneration of muscle fibres was slow, probably because freezing had killed many satellite cells in the muscle. Because the ChAT activity at day 12 had recovered when CK was almost absent and the preparation failed to contract, we conclude that there was expression of ChAT activity in ‘nerve terminals’ which do not make contact with regenerated muscle cells, although little if any ACh was released from these sites. ©1998 Elsevier Science Ltd. All rights reserved.     

Neuromechanical effects of pyrethroids, allethrin, cyhalothrin and deltamethrin on the cholinergic processes in rat brain

Our previous microdialysis study of freely moving rats demonstrated that 3 pyrethroids, allethrin (type I), cyhalothrin (type II) and deltamethrin (type II) differentially modulate acetylcholine (ACh) release in the hippocampus. To better understand the mechanisms of their modulatory effects and also other effects on the cholinergic system in the brain, the activities of ACh hydrolyzing enzyme acetylcholinesterase (AChE), ACh synthesizing enzyme choline acetyltransferase (ChAT) and ACh synthesizing rate-limiting step, high-affinity choline uptake (HACU) were examined in the present study. The pyrethroids studied had no effect on AChE activity in the cortex, hippocampus and striatum. These pyrethroids had no significant effect on ChAT in the cortex and hippocampus, but striatal ChAT was increased at higher dosage (60 mg/kg) by all three compounds. Lineweaver-Burk analysis of hippocampal HACU revealed that the pyrethroids did not alter the Michaelis-Menten constant (Km) value but caused alteration of maximal velocity (Vmax). Allethrin (60 mg/kg) and cyhalothrin (20 and 60 mg/kg) decreased while deltamethrin (60 mg/kg) increased the Vmax for HACU. In vitro study showed that at higher concentrations (> or = 10(-) (6) M) allethrin and cyhalothrin reduced the hippocampal HACU but deltamethrin increased it. These results suggest that mechanisms of ACh synthesis are involved in the modulatory effects of the pyrethroids on ACh release and other cholinergic activities.     

Effects of Chronic Basic Fibroblast Growth Factor Administration to Rats with Partial Flmbrial Transections on Presynaptic Cholinergic Parameters and Muscarinic Receptors in the Hippocampus: Comparison with Nerve Growth Factor

Abstract: The present study compares the effects of chronic administration of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on various hippocampal cholinergic parameters in rats with partial unilateral fimbrial transections. Lesions resulted in marked reductions of several presynaptic cholinergic parameters: choline acetyltransferase (ChAT) activity (by 50%), [³H]-acetylcholine ([³H]ACh) synthesis (by 59%), basal and ve-ratridine (1 μM)-evoked [³H]ACh release (by 44 and 57%, respectively), and [³H]vesamicol binding site densities (by 35%). In addition, [³H]AF-DX 116/muscarinic M₂ binding site densities were also modestly decreased (by 23%). In contrast, [³H]pirenzepine/muscarinic M₁ and [³H]AF-DX 384/muscarinic M₂/M₄ binding site densities were not altered by the lesions, nor were they affected by any of the treatments. Intracerebroventricular administration of bFGF (10 ng, every other day, for 21 days) partially prevented the lesion-induced deficit in hippocampal ChAT activity, an effect that was not markedly different from that measured in the NGF-treated (1 μg intracerebroventricularly, every other day, for 21 days) rats. In rats treated with a combination of bFGF and NGF, ChAT activity was not different from that in rats treated with the individual factors alone. In contrast, the lesion-induced deficits in the other cholinergic parameters were not attenuated by bFGF treatment, although they were at least partially prevented by NGF administration. To determine whether higher concentrations of bFGF are necessary to affect cholinergic parameters other than hippocampal ChAT activity, rats were treated with 1 μg (every other day, 21 days) of the growth factor. In this group of rats, detrimental effects of bFGF, manifested by an increased death rate (46%), and marked reductions in body weight of the survivors, were observed. In addition, this concentration of bFGF appeared to exacerbate the lesion-induced reduction in [³H]ACh synthesis by hippocampal slices; [³H]ACh synthesis in lesioned hippocampi represented 36 and 52% of that in contralateral unlesioned hippocampi for the bFGF-treated and control groups, respectively. In conclusion, although bFGF administration attenuates the deficit in hippocampal ChAT activity induced by partial fimbrial transections, this does not appear to translate into enhanced functional capacity of the cholinergic terminals. This is clearly in contrast to NGF, which enhances not only hippocampal ChAT activity, but also other parameters indicative of increased function in the cholinergic terminals.     

Stimulation by GH of IGF1 proforms synthesized by rabbit chondrocytes cultured with bFGF in serum-free medium

The IR-IGF1 production by rabbit epiphyseal chondrocytes cultured in serum-free medium was analyzed. Cell proliferation was induced by the addition of 10 ng/ ml basic fibroblast growth factor (bFGF) without or with 100 ng/ml recombinant human growth hormone (hGH). GH alone induced no cell multiplication. Chondrocytes treated with bFGF alone secreted an IR-IGF1 activity proportional to the mitotic activity of the cells. A specific positive IGF1 immunostaining was localized in the Golgi of control and hGH-treated cells. The IR-IGF1 activity recovered into culture medium was mainly composed of three fractions of apparent MW 6-8 kDa, 9–14 kDa, and 16–18 kDa. [³⁵S]Methionine pulse-chase experiments indicated that the radiolabeled 16–18 kDa IR-IGF1 fraction was partly converted into the 9–14 kDa and 6–8 kDa fractions. At equilibrium, 70% of the chondrocyte IR-IGF1 activity was recovered as 9- to 18-kDa forms which contained high IR-proIGF1A activity. The 6–8 kDa fraction had biochemical characteristics similar to those of the mature IGF1 peptide. Similar results were observed when 4% fetal calf serum was added to the culture. The addition of 100 ng/ml of hGH significantly and specifically increased IGF1 precursor material, which thus represented 90% of total IR-IGF1 activity. On Day 16 of the culture, when cells stopped dividing, the amount of chondrocyte IR-IGF1 was significantly lower than during cell proliferation, and hGH had no effect on this production. These data indicate that cultured chondrocytes produce more IGF1 precursors than mature IGF1 and that GH specifically stimulates biosynthesis of IGF1 precursors but not IGF1 per se. A GH-dependent biological function of IGF 1 preforms in chondrocytes remains to be demonstrated.     

Functional Regulation of Choline Acetyltransferase by Phosphorylation

Choline acetyltransferase (ChAT) catalyzes synthesis of acetylcholine (ACh) in cholinergic neurons. ACh synthesis is regulated by availability of precursors choline and acetyl coenzyme A or by activity of ChAT; ChAT regulates ACh synthesis under some conditions. Posttranslational phosphorylation is a common mechanism for regulating the function of proteins. Analysis of the primary sequence of 69-kD human ChAT indicates that it has putative phosphorylation consensus sequences for multiple protein kinases. ChAT is phosphorylated on serine-440 and threonine-456 by protein kinase C and CaM kinase II, respectively. These phosphorylation events regulate activity of the enzyme, as well as its binding to plasma membrane and interaction with other cellular proteins. It is relevant to investigate differences in constitutive and inducible patterns of phosphorylation of ChAT under physiological conditions and in response to challenges that cholinergic neurons may be exposed to, and to determine how changes in phosphorylation relate to changes in neurochemical transmission.     

Acetyl-CoA Synthesizing Enzymes in Cholinergic Nerve Terminals

The activities of five enzymes involved in acetyl-CoA synthesis, pyruvate dehydrogenase complex, ATP citrate lyase, carnitine acetyltransferase, acetyl-CoA synthetase, and citrate synthase, were determined in normal nucleus interpeduncularis and nucleus interpeduncularis in which cholinergic terminals were removed following lesion of the habenulointerpeduncular tract. The activities of aspartate transaminase, fumarase, and GABA transaminase also were determined to compare the effect of lesion on other mitochondrial enzymes which are not linked to the biosynthesis of ACh. In normal nucleus interpeduncularis the activities of carnitine acetyltransferase and pyruvate dehydrogenase complex were higher than the activity of ChAT (choline acetyltransferase), whereas the activities of acetyl-CoA synthetase and citrate synthase were considerably lower than that of ChAT. The effect of the lesion separated the enzymes into two groups: the activities of pyruvate dehydrogenase complex, carnitine acetyltransferase, fumarase and aspartate transaminase decreased by 30--40%, whereas the activities of the other enzymes descreased 5--15%. ChAT activity was in all cases less than 15% of normal. It could be concluded that none of the acetyl-CoA synthesizing enzymes decreased to the degree that ChAT did. Only pyruvate dehydrogenase complex and carnitine acetyltransferase seem to be localized in cholinergic terminals to a significant degree. ATP citrate lyase as well as acetyl-CoA synthetase seem to have less significance in supporting acetyl-CoA formation in cholinergic nerve terminals.     

Soluble and membrane-associated heat shock proteins in soybean root

Summary Localization of heat shock proteins (Hsp) in endomembranes and determination of whether they are integral or peripheral membrane proteins will aid in understanding the physiological function of the heat shock response. Radiolabeled endomembranes (endoplasmic reticulum, Golgi, and plasma membrane), obtained by sucrose gradient centrifugation of heat-shocked soybean (Glycine max L.) root tissue were solubilized and the polypeptides separated by two-dimensional IEF-SDS-PAGE. Autoradiography revealed three groups of Hsp. A diverse group fo 25 low mol wt Hsp (18 to 24 kDa) with isoelectric point (pI) between 5 and 7; an intermediate mol wt group (30 to 47 kDa) with pI of 5.5 to 6.0; and a group of two high mol wt Hsp (75 to 80 kDa) with pI 4.8 to 5.2. The plasma membrane fraction lacked the Hsp pair of 47 kDa detected in the endoplasmic reticulum and Golgi fractions but possessed a unique Hsp of 30 kDa, pI 5.5.Comparison of soluble and microsome fractions revealed a difference in the pattern of the low mol wt Hsp class. The soluble fraction contained Hsp of 16–20 kDa with pI between 5 and 7.8 while the microsome fraction was characterized by Hsp of 18–24 kDa with pI between 5.8 and 6.5.The microsomal Hsp were not released by 1 M KCl. Treatment of the microsome fraction with Triton X-100 selectively released several Hsp, and Na₂CO₃ treatment removed additional Hsp from the membrane fraction.Abbreviations Hsp heat-shock protein(s) - GA Golgi apparatus - PM plasma membrane - 2 D two-dimensional     

Immunological,isoelectric, hydrophobic and molecular weight differences between soluble and ionically membrane-bound fractions of choline-o-acetyltransferase prepared from mouse and rat brain

Choline-O-acetyltransferase (EC 2.3.1.6; ChAT) was prepared from synaptosomal fractions (P₂) of mouse and rat brain in the presence of proteolytic inhibitors by the method of Gray and Whittaker (1962) as modified by (Salehmoghaddam and Collier, 1976). The P₂ fraction was hypo-osmotically shocked with glass distilled water and centrifuged to separate the cytoplasmic (S₃) and vesicle-bound (P₃) fractions. Fraction S₃ was saved for ChAT assay and compared with the ChAT fraction eluted from the P₃ by salt at a pH 7.4 or by detergent (Benishin and Carroll, 1983). These three fractions of ChAT were then compared by molecular weights, isoelectric points, immunoblotting with monoclonal or polyclonal antibodies and hydrophobicity. The results show that the S₃ fraction of ChAT has a molecular weight of 66 K_d, whereas the ionically-bound fraction of ChAT has a molecular weight of 73–78 K_d. SDS-PAGE of these two ChAT fractions followed by immunoblotting revealed the presence of two immunoreactive bands at 28–29 K_d and 50–51 K_d for the ionically bound ChAT fraction. Conversely, none of these antibodies immunostained any protein bands for the S₃ ChAT fraction even though one monoclonal antibody had been prepared against this ChAT fraction and the S₃ ChAT fraction had a similar specific activity prior to SDS-PAGE as did the salt solubilized ChAT fraction. However, anti-ChAT monoclonal antibody MB16 binds the native S₃ ChAT fraction in the co-precipitation assay.The S₃ fraction of ChAT had only one isoelectric point at pH 7.8, whereas the ionically bound and detergent soluble ChAT fractions had two isoelectric points at pH 8.1–8.15 and 7.45–7.5. The S₃ ChAT fraction also differed in hydrophobicity from the other two ChAT fractions. These differences between the S₃ and salt soluble ChAT fractions were not obviated by addition of Triton X-100 and thus could not be attributed to the association of lipids with either of the fractions. We conclude that the water soluble fraction of ChAT in central nerve terminals differs in its physical properties and its subcellular location from that which ionically binds to membranes.     

The lymphocytic cholinergic system and its biological function

Lymphocytes are now known to possess the essential components for a non-neuronal cholinergic system. These include acetylcholine (ACh); choline acetyltransferase (ChAT), its synthesizing enzyme; and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Stimulating lymphocytes with phytohemagglutinin, a T-cell activator; Staphylococcus aureus Cowan I, a B-cell activator; or cell surface molecules enhances the synthesis and release of ACh and up-regulates expression of ChAT and M(5) mAChR mRNAs. Activation of mAChRs and nAChRs on lymphocytes elicits increases in the intracellular Ca(2+) concentration and stimulates c-fos gene expression and nitric oxide synthesis. On the other hand, long-term exposure to nicotine down-regulates expression of nAChR mRNA. Abnormalities in the lymphocytic cholinergic system have been detected in spontaneously hypertensive rats and MRL-lpr mice, two animal models of immune disorders. Taken together, these data present a compelling picture in which immune function is, at least in part, under the control of an independent non-neuronal lymphocytic cholinergic system.     

A Non-Neuronal Cardiac Cholinergic System Plays a Protective Role in Myocardium Salvage during Ischemic Insults

Background

In our previous study, we established the novel concept of a non-neuronal cardiac cholinergic system–cardiomyocytes produce ACh in an autocrine and/or paracrine manner. Subsequently, we determined the biological significance of this system–it played a critical role in modulating mitochondrial oxygen consumption. However, its detailed mechanisms and clinical implications have not been fully investigated.

Aim

We investigated if this non-neuronal cardiac cholinergic system was upregulated by a modality other than drugs and if the activation of the system contributes to favorable outcomes.

Results

Choline acetyltransferase knockout (ChAT KO) cells with the lowest cellular ACh levels consumed more oxygen and had increased MTT activity and lower cellular ATP levels compared with the control cells. Cardiac ChAT KO cells with diminished connexin 43 expression formed poor cell–cell communication, evidenced by the blunted dye transfer. Similarly, the ChAT inhibitor hemicholinium-3 decreased ATP levels and increased MTT activity in cardiomyocytes. In the presence of a hypoxia mimetic, ChAT KO viability was reduced. Norepinephrine dose-dependently caused cardiac ChAT KO cell death associated with increased ROS production. In in vivo studies, protein expression of ChAT and the choline transporter CHT1 in the hindlimb were enhanced after ischemia-reperfusion compared with the contralateral non-treated limb. This local effect also remotely influenced the heart to upregulate ChAT and CHT1 expression as well as ACh and ATP levels in the heart compared with the baseline levels, and more intact cardiomyocytes were spared by this remote effect as evidenced by reduced infarction size. In contrast, the upregulated parameters were abrogated by hemicholinium-3.

Conclusion

The non-neuronal cholinergic system plays a protective role in both myocardial cells and the entire heart by conserving ATP levels and inhibiting oxygen consumption. Activation of this non-neuronal cardiac cholinergic system by a physiotherapeutic modality may underlie cardioprotection through the remote effect of hindlimb ischemia-reperfusion.     

Selective Expression of Factors Preventing Cholinergic Dedifferentiation

Chicken retina neurons from 8-9-day-old embryos developed prominent cholinergic properties after several days in stationary dispersed cell (monolayer) culture. These cells accumulated [3H]choline by a high-affinity, hemicholinium-sensitive transport system, converted [3H]choline to [3H]-acetylcholine [( 3H]ACh), released [3H]ACh in response to depolarization stimuli, and developed choline acetyltransferase (ChAT) activity to levels comparable to those of the intact retina. The cholinergic state, however, was not permanent. After 7 days in culture, the capacity for [3H]ACh release decreased drastically and continued to diminish with longer culture periods. Loss of this capacity seemed not to be due to loss of cholinergic neurons, because high-affinity choline uptake was unchanged. However, a substantial decrease of ChAT activity was observed as a function of culture age, and probably accounted for the low level of ACh synthesis in long-lasting cultures. The loss of ChAT activity could be prevented in at least two different ways: (a) Maintaining the neurons in rotary (aggregate) rather than stationary culture completely blocked the loss of enzyme activity and gave a developmental profile identical to the known "in situ" pattern of differentiation; and (b) Conditioned medium from aggregate cultures significantly reduced the drop in ChAT activity of neurons maintained in stationary, dispersed cell cultures. Activity that stabilized cholinergic differentiation was nondialyzable, heat-sensitive, and not mimicked by functional nerve growth factor. Production of activity by aggregates was developmentally regulated; medium obtained from aggregates after 3 days in culture had no effect on cholinergic differentiation, whereas medium obtained from aggregates between 6 and 10 days in culture produced a fivefold increase of ChAT in monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of Alzheimer's ß-Amyloid Protein by Human and Rat Brain Peptidases: Involvement of Insulin-Degrading Enzyme

We examined the degradation of Alzheimer's ß-amyloid protein (1–40) by soluble and synaptic membrane fractions from post mortem human and fresh rat brain using HPLC. Most of the activity at neutral pH was in the soluble fraction. The activity was thiol and metal dependent, with a similar inhibition profile to insulin-degrading enzyme. Immunoprecipitation of insulin-degrading enzyme from the human soluble fraction using a monoclonal antibody removed over 85% of the ß-amyloid protein degrading activity. Thus insulin-degrading enzyme is the main soluble ß-amyloid degrading enzyme at neutral pH in human brain. The highest ß-amyloid protein degrading activity in the soluble fractions occurred between pH 4–5, and this activity was inhibited by pepstatin, implicating an aspartyl protease. Synaptic membranes had much lower ß-amyloid protein degrading activity than the soluble fraction. EDTA (2mM) caused over 85% inhibition of the degrading activity but inhibitors of endopeptidases –24.11, –24.15, –24.16, angiotensin converting enzyme, aminopeptidases, and carboxypeptidases had little or no effect.     

ATP-induced currents in submucous plexus neurons of the guinea pig small intestine

ATP-induced membrane durrents in the submucous neurons of the guinea pig small intestine were studied using the whole-cell patch-clamp recording technique. Being applied at –50 mV. ATP activated an inward non-selective cationic current in 68.3% of the investigated neurons. An increase in ATP concentration within the 1–1,000 µM range resulted in the s-like increase in the amplitude of ATP-induced current. The EC₅₀ was 150.0±18.5 µM, while the Hill number was 1.6. The current was selectively activated by ATP and was not blocked by P2 purinoreceptor antagonist suramin (50–300 µM).,-Methylene-ATP (100–200 µM) and,-methylene-ATP (100–200µM), which are P2-purinoreceptor agonists, as well as adenosine (100–300 µM), exerted no effects. Reactive blue 2, if applied up to 4 min, enhanced ATP-induced current, while its longer application partially suppressed this current. In most submucous neurons, acetylcholine (ACh) likewise activated an inward cationic current. The amplitude of ACh-induced current was lower if ACh was applied during a long-lasting application of ATP than if ACh only was applied. Hexamethonium (50 µM), d-tubocurarine (20–40 µM), and trimethaphan (30 µM) completely and reversibly blocked ACh-induced currents, regardless of the presence of ATP, and did not affect ATP-induced currents. The results suggest that ATP-induced currents in submucous neurons are due to activation of a unique type of P2 purinoreceptors, which function in connection with nicotinic ACh receptors.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 100–110, March–June, 1996.     

Separation and partial purification of extracellular amylase and protease from Bacillus caldolyticus

Summary The extracellular amylase and protease from Bacillus caldolyticus can be concentrated by ammonium sulfate precipitation after growth on either solid or in liquid media containing starch, glucose, and brain-heart infusion. Using the Diaflo ultrafiltration system with membranes of various permeability, the enzymes could be separated from each other by extensive flushing with buffer. Best results were obtained with the 50–70% ammonium sulfate fraction as starting material, yielding 72% of the total amylase activity in the low molecular weight fraction (UM-10 fraction: 10000–30000), while 54 and 25% respectively of the protease were retained in the two high molecular weight fractions (50000–100000, and more than 100000). Similar results were obtained with the 20–50% ammonium sulfate fraction, while the fraction of 0–20% saturation contained a low molecular weight protease. The native amylase seems to consist of a number of sub-units, which after extensive flushing accumulate in the fraction with an approximate molecular weight between 10000 and 30000. The enzyme could also be precipitated from cell-free liquid media with ammonium sulfate, followed by separation and purification on ultra-filtration cells. According to the specific activity of the UM-10 fractions a 400-fold purification was obtained compared to the amylase activity of the cell-free medium.Direct concentration and separation from liquid media, omitting ammonium sulfate treatment, was also found to be possible, although prolonged flushing with buffer was necessary to obtain satisfactory separation.During purification from the ammonium sulfate fractions, amylase activity was found to decrease but could be restored by Ca-ions. At 70°C, a final concentration of 0.5 mM CaCl₂, was sufficient for full restoration, while three times that amount was necessary at 80°C. Determination of the K _m-values for Ca at different temperatures resulted in an asymptotically increasing curve at temperatures beyond 75°C. Addition of Ca had a pronounced effect on the stability of the amylase at 80°C but not at 90°C. Protease activity and stability was not affected by Ca-ions.     

Development of NMR spectroscopic methods for dynamic detection of acetylcholine synthesis by choline acetyltransferase in hippocampal tissue

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post‐mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used ¹⁵N‐choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before ¹H‐NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT‐mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real‐time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non‐invasive manner using NMR spectroscopic techniques.