The potential ability of Al to affect cholinergic transmission was studied on synaptosomal fractions of rat brain incubated with pyruvate in depolarizing medium containing 30 mM K+. Addition of 1 mM Ca caused a 266% increase in the acetylcholine (ACh) release despite decreased pyruvate oxidation. Under these conditions, 0.25 mM Al did not affect pyruvate oxidation but raised mitochondrial and decreased synaptoplasmic acetyl‐CoA. Simultaneously, a 61% inhibition of Ca‐evoked ACh release was observed. Verapamil (0.1 and 0.5 mM) decreased the acetyl‐CoA concentration in synaptoplasm and inhibited ACh release. Al (0.012 mM) partially reversed these inhibitory effects. Omission of Pi from the medium abolished suppressive effects of Al on acetyl‐CoA content and Ca‐evoked transmitter release. We conclude that the Al(PO4)OH− complex may be the active form of Al, which, by interaction with the verapamil binding sites of Ca channels, is likely to restrict the Ca influx to the synaptoplasm. This may inhibit the provision of acetyl‐CoA to the synaptoplasm as well as the Ca‐evoked ACh release. One may suppose that excessive accumulation of Al in some encephalopathic brains may, by this mechanism, suppress still‐surviving cholinergic neurons and exacerbate cognitive deficits caused by already‐existing structural losses in the cholinergic system.

Nerve growth factor (NGF) is a peptide displaying multiple cholinotropic activities. The aim of this work was to explain mechanisms of the positive and negative effects of NGF on phenotypic properties and viability of cholinergic cells. To discriminate these effects we used two p75 NTR receptor-pos- Nerve growth factor (NGF) is one of the neurotrophins with distinct cholinotropic activity in the brain. It was found to increase activities of choline acetyltransferase (choline-Oacetyltransferase, ChAT, EC 2.3.1.6) and vesicular acetylcholine transporter, the acetylcholine (ACh) level and rate of its release/synthesis as well as density of cholinergic M2 autoreceptors in septum, hippocampus and other cortical areas containing cholinergic neurons and their axonal terminals (Tonnaer et al. 1994). NGF resulted in regeneration of transsected cholinergic septo-hippocampal fibres (Horner and Gage 2000). Similar cholinotrophic effects of NGF were observed in primary and clonal cultures of cholinergic cells where it brought about increase of expression of mRNA, protein and activity of ChAT along with elevation of ACh content and release (Oosawa et al. 1999). These effects were mediated by activation of specific, high affinity NGF TrkA receptors autophosphorylated at tyrosine residues found on the cytoplasmic domain with subsequent activation of multiple signal transduction pathways. Cooperation of the p75 NTR low affinity, non-specific receptor with the TrkA receptor plays an important role in modulation of NGF Received January 28, 2004; revised manuscript received March 10, 2004; accepted April 20, 2004. Address corespondence and reprint requests to Andrzej Szutowicz, Chair of Clinical Biochemistry, Department of Laboratory Medicine, Medical University of Gdañsk, Dêbinki 7 Str., 80-211 Gdañsk, Poland. E-mail: aszut@amg.gda.plAbbreviations used: Ab, amyloid b-peptide; ACh, acetylcholine; ChAT, choline acetyltransferase; CREB, cAMP response element binding protein; DC, cells diffrentiated by cAMP and retinoic acid; NC, nondifferentiated cells; NGF, b-nerve growth factor; NO, nitric oxide; p75 NTR , low affinity neurotrophic receptor; PDH, pyruvate dehydrogenase; RA, all-trans-retinoic acid; SNP, sodium nitroprusside; TrkA, high affinity nerve growth factor receptor.

Cholinergic neurons, unlike other brain cells utilize acetyl-CoA not only for energy production but also for acetylcholine (ACh) synthesis. Therefore, suppression of acetyl-CoA metabolism by different neurotoxic inputs may be particularly harmful for this group of cells. Differentiation of SN56 cholinergic hybrid cells increased their choline acetyltransferase (ChAT) activity and ACh content but depressed pyruvate dehydrogenase activity and acetyl-CoA content. Differentiated cells were more susceptible to acute and chronic influences of aluminum, NO and amyloid-beta. Al decreased acetyl-CoA content, ACh release and increased Ca accumulation in differentiated cells (DC) to much higher degree than in non-differentiated ones (NC). NO strongly depressed acetyl-CoA level and increased ACh release in DC but did not affect NC. Additive effects of Al and NO were seen in DC but not in NC. Also long term suppressory effects of amyloid-beta, Al and NO on cholinergic phenotype and morphologic maturation were more evident in DC than in NC. Thus, relative shortage of acetyl-CoA in highly differentiated cholinergic neurons could make them particularly susceptible to degenerative insults in the course of different cholinergic encephalopathies.

Different groups of brain cholinergic neurons display variable susceptibility to similar neurotoxic inputs. The aim of this work was to find out whether changes in cholinergic phenotype may alter the availability of acetyl-CoA in mitochondrial compartment and thereby the viability of cholinergic neurons. Cyclic AMP (cAMP) and retinoic acid caused differentiation (DC) of T17 TrkA(+) cholinergic neuroblastoma cells. In addition, it increased the choline acetyltransferase (ChAT) activity, Ca(2+) accumulation and cytoplasmic acetyl-CoA level, but decreased mitochondrial acetyl-CoA and cell resistance to amyloid-beta(25-35) (Abeta) toxicity. Nerve growth factor (NGF) caused similar alterations in the nondifferentiated cells (NC). On the other hand, in DC NGF suppressed ChAT activity and elevated mitochondrial level of acetyl-CoA but also caused a further increase of Ca(2+) content and cell susceptibility to Abeta. The significant inverse correlation was found between ChAT activity and mitochondrial levels of acetyl-CoA. Abeta markedly reduced the expression of cholinergic phenotype, acetyl-CoA content, and viability of DC. These effects were absent or much less pronounced in NC. Acetyl-L-carnitine reversed suppressing effects of Abeta on acetyl-CoA levels and ChAT activity but did not reverse increased mortality in DC. Presented data indicate that increased transmitter activity in highly differentiated cholinergic neurons, decreased acetyl-CoA level in their mitochondrial compartment, and increased Ca(2+) accumulation can make them more prone to neurotoxic conditions. Phenotype-dependent changes in intracellular distribution of acetyl-CoA thus play an important role in regulation of viability and transmitter function in brain cholinergic neurons.

Acetylcholine and acetyl-CoA metabolism in nerve terminals isolated from rat brain were found to be affected by several neurotoxic and neuroprotective agents, such as aluminium, nitric oxide, β-hydroxybutyrate, verapamil and thiamine deficiency. The changes evoked by these factors in Ca²⁺-dependent acetylcholine release were highly significantly correlated (r = 0.98) with changes in concentration of synaptoplasmic acetyl-CoA. On the other hand, in the same experimental conditions, no correlation was found between rates of pyruvate oxidation, intramitochondrial acetyl-CoA levels and different pools of releasable acetylcholine. These data indicate that disturbances in the availability of acetyl-CoA in the cytoplasm of nerve terminals may be a key factor in the pathogenesis of several cholinergic encephalopathies.