Karoly Gulya

The cholinergic system in alzheimer's disease

The past decade has witnessed an enormous increase in our knowledge of the variety and complexity of neuropathological and neurochemical changes in Alzheimers disease. Although the disease is characterized by multiple deficits of neurotransmitters in the brain, this overview emphasizes the structural and neurochemical localization of the elements of the acetylcholine system (choline acetyltransferase, acetylcholinesterase, and muscarinic and nicotinic acetylcholine receptors) in the non-demented brain and in Alzheimers disease brain samples. The results demonstrate a great variation in the distribution of acetylcholinesterase, choline acetyltransferase, and the nicotinic and muscarinic acetylcholine receptors in the different brain areas, nuclei and subnuclei. When stratification is present in certain brain regions (olfactory bulb, cortex, hippocampus, etc.), differences can be detected as regards the laminar distribution of the elements of the acetylcholine system. Alzheimers disease involves a substantial loss of the elements of the cholinergic system. There is evidence that the most affected areas include the cortex, the entorhinal area, the hippocampus, the ventral striatum and the basal part of the forebrain. Other brain areas are less affected. The fact that the acetylcholine system, which plays a significant role in the memory function, is seriously impaired in Alzheimers disease has accelerated work on the development of new drugs for treatment of the disease of the 20th century.

Brain regional specificity and time-course of changes in the NMDA receptor-ionophore complex during ethanol withdrawal

Previous work, using membrane receptor binding techniques, demonstrated an increase in hippocampal MK-801 binding sites in mice after chronic ethanol ingestion. The current studies, using quantitative autoradiography, demonstrate that chronic ethanol ingestion also produces increases in MK-801 binding in cerebral cortex, striatum and thalamus, as well as in hippocampus. The persistence of changes in MK-801 binding paralleled the time-course for ethanol withdrawal seizure susceptibility. These results support the hypothesis that an increase in the number of NMDA receptor/channel complexes in hippocampus, and possibly other brain regions, plays a role in the generation or expression of ethanol withdrawal seizures.

Chronic ethanol ingestion decreases vasopressin mRNA in hypothalamic and extrahypothalamic nuclei of mouse brain.

Endogenous arginine vasopressin was previously shown to modulate the rate of loss of functional (CNS) tolerance to ethanol, suggesting that chronic ethanol ingestion might alter vasopressin synthesis and/or release. Since extrahypothalamic vasopressin is believed to be involved in the CNS effects of the peptide, we determined the effect of ethanol on vasopressin mRNA in the bed nucleus of the stria terminalis (BST), as well as in several hypothalamic nuclei. Chronic ethanol ingestion, that produced functional tolerance and physical dependence in mice, resulted in decreased vasopressin mRNA levels in all areas examined. In contrast, as expected, dehydration resulted in increases in vasopressin mRNA in the BST and in all hypothalamic nuclei except the suprachiasmatic nucleus. In the BST, both ethanol ingestion and dehydration affected cells in the central region of the nucleus, while cells in the caudal portion were only affected by ethanol treatment. The results indicate that chronic ethanol ingestion generally reduces the synthesis of vasopressin, and that increased vasopressin synthesis is not necessary in order for the peptide to affect ethanol tolerance.

Cyclic somatostatin octapeptide analogues with high affinity and selectivity toward mu opioid receptors

A series of cyclic conformationally restricted penicillamine containing somatostatin octapeptide analogues have been prepared by standard solid phase synthetic techniques and tested for their ability to inhibit specific [125I]CGP 23,996 (des-Ala1-,Gly2-[desamino-Cys3Tyr11]-dicarba3, 14-somatostatin), [3H]naloxone or [3H]DPDPE ([D-Pen2-D-Pen5]enkephalin) binding in rat brain membrane preparations. We now report structure-activity relationship studies with the synthesis of our most potent and selective mu opioid receptor compound D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, which we refer to as Cys2Tyr3Orn5Pen7-amide. While this octapeptide exhibited high affinity (IC50 = 2.80 nM) for an apparently single population of binding sites (nH = 0.89 +/- 0.1) and exceptional selectivity for mu opioid receptors with an IC50(DPDPE)/IC50 (naloxone) ratio of 4,829, it also displayed very low affinity for somatostatin receptors (IC50 = 22,700 nM). Thus, Cys2Tyr3Orn5Pen7-amide may be the ligand of choice for further characterization of mu opioid receptors and for examining the physiological role of this class of receptors.

Cholinotoxic Effects of Aluminum in Rat Brain

Abstract: The in vivo and in vitro effects of A1 on the cholinergic system of rat brain were studied. The amount of A1 accumulated after the chronic, intraperitoneal administration of aluminum gluconate (Al‐G) or AlCl3, both at a dose of 1 mg/ml/100 g of body weight, increased in the frontal and parietal cortices, the hippocampus, and the striatum. Significantly decreased choline acetyltransferase activities after chronic Al treatment were measured in the parietal cortex, the hippocampus, and the striatum, but not in the frontal cortex. The acetylcholinesterase activity was not changed significantly in any brain area investigated. Both Al‐G and AlCl3 administrations resulted in a general decrease (to 40–70% of the control values) in the specific l‐[3H]nicotine binding, involving all brain areas studied. The specific (–)‐[3H]quinuclidinyl benzilate binding was reduced (to 40–60% of the control values) only after 25 days of Al treatment. Al‐G and AlCl3 were equivalent in eliciting these reductions. In vitro studies revealed different alterations of the cholinergic system in response to Al treatment. No changes were observed either in choline acetyltransferase activity or in cholinergic receptor bindings. Both Al‐G and Al2(SO4)3 treatments, however, exhibited a biphasic effect on the acetylcholinesterase activity. At low Al concentrations (10–8–10–6M), the activity was slightly increased, whereas at higher concentrations (10–6–10–4M), it was inhibited by a maximum of 25% as compared to the controls. Thus, these cholinotoxic effects are probably due not to a direct interaction between the metal and the cholinergic marker proteins, but rather to a manifestation and consequence of its neurodegenerative effects.

Activated MAO-B in the brain of Alzheimer patients, demonstrated by [11C]-L-deprenyl using whole hemisphere autoradiography

In the human brain the monoaminooxidase-B enzyme or MAO-B is highly abundant in astrocytes. As astrocyte activity and, consequently, the activity of the MAO-B enzyme, is up-regulated in neuroinflammatory processes, radiolabelled analogues of deprenyl may serve as an imaging biomarker in neuroinflammation and neurodegeneration, including Alzheimers disease. In the present study [(11)C]-L-deprenyl, the PET radioligand version of L-deprenyl or selegiline®, a selective irreversible MAO-B inhibitor was used in whole hemisphere autoradiographic experiments in human brain sections in order to test the radioligands binding to the MAO-B enzyme in human brain tissue, with an eye on exploring the radioligands applicability as a molecular imaging biomarker in human PET studies, with special regard to diagnostic detection of reactive astrogliosis. Whole hemisphere brain sections obtained from Alzheimer patients and from age matched control subjects were examined. In control brains the binding of [(11)C]-L-deprenyl was the highest in the hippocampus, in the basal ganglia, the thalamus, the substantia nigra, the corpus geniculatum laterale, the nucleus accumbens and the periventricular grey matter. In Alzheimer brains significantly higher binding was observed in the temporal lobes and the white matter. Furthermore, in the Alzheimer brains in the hippocampus, temporal lobe and white matter the binding negatively correlated with Braak stages. The highest binding was observed in Braak I-II, whereas it decreased with increasing Braak grades. The increased regional binding in Alzheimer brains coincided with the presence of an increased number of activated astrocytes, as demonstrated by correlative immunohistochemical studies with GFAP in adjacent brain slices. Deprenyl itself as well as the MAO-B antagonist rasagiline did effectively block the binding of the radioligand, whereas the MAO-A antagonist pirlindole did not affect it. Compounds with high affinity for the PBR system did not block the radioligand binding either, providing evidence for the specificity of [(11)C]-L-deprenyl for the MAO-B enzyme. In conclusion, the present observations indicate that [(11)C]-L-deprenyl may be a promising and selective imaging biomarker of increased MAO-B activity in the human brain and can therefore serve as a prospective PET tracer targeting neuroinflammation and neurodegeneration.

Cholinotoxic effects of β-amyloid(1–42) peptide on cortical projections of the rat nucleus basalis magnocellularis

Beta-amyloid(1-42) peptide (betaAP) was injected into the right nucleus basalis magnocellularis (nbm) of rats. After a 14-day survival time, the acetylcholinesterase and choline acetyltransferase activities and the number of muscarinic receptors were found biochemically to be significantly reduced in the ipsilateral frontal cortices. Confirmation of these data with silver staining also revealed degeneration of the projective fibers of the nbm to the frontal cortex. These results demonstrate the cholinotoxicity of betaAP in an in vivo animal model.

A comparative autoradiography study in post mortem whole hemisphere human brain slices taken from Alzheimer patients and age-matched controls using two radiolabelled DAA1106 analogues with high affinity to the peripheral benzodiazepine receptor (PBR) system

The binding of two radiolabelled analogues (N-(5-[125I]Iodo-2-phenoxyphenyl)-N-(2,5-dimethoxybenzyl)acetamide ([125I]desfluoro-DAA1106) and N-(5-[125I]Fluoro-2-phenoxyphenyl)-N-(2-[125I]Iodo-5-methoxybenzyl)acetamide ([125I]desmethoxy-DAA1106) of the peripheral benzodiazepine receptor (PBR) (or TSPO, 18kDa translocator protein) ligand DAA1106 was examined by in vitro autoradiography on human post mortem whole hemisphere brain slices obtained from Alzheimers disease (AD) patients and age-matched controls. Both [(125)I]desfluoro-IDAA1106 and [(125)I]desmethoxy-IDAA1106 were effectively binding to various brain structures. The binding could be blocked by the unlabelled ligand as well as by other PBR specific ligands. With both radiolabelled compounds, the binding showed regional inhomogeneity and the specific binding values proved to be the highest in the hippocampus, temporal and parietal cortex, the basal ganglia and thalamus in the AD brains. Compared with age-matched control brains, specific binding in several brain structures (temporal and parietal lobes, thalamus and white matter) in Alzheimer brains was significantly higher, indicating that the radioligands can effectively label-activated microglia and the up-regulated PBR/TSPO system in AD. Complementary immunohistochemical studies demonstrated reactive microglia activation in the AD brain tissue and indicated that increased ligand binding coincides with increased regional microglia activation due to neuroinflammation. These investigations yield further support to the PBR/TSPO binding capacity of DAA1106 in human brain tissue, demonstrate the effective usefulness of its radio-iodinated analogues as imaging biomarkers in post mortem human studies, and indicate that its radiolabelled analogues, labelled with short half-time bioisotopes, can serve as prospective in vivo imaging biomarkers of activated microglia and the up-regulated PBR/TSPO system in the human brain.

[3H]AF-DX 116 labels subsets of muscarinic cholinergic receptors in rat brain and heart☆

The in vitro binding properties of the novel muscarinic antagonist [3H]AF-DX 116 were studied using a rapid filtration technique. Association and dissociation rates of [3H]AF-DX 116 binding were rapid at 25 degrees C (2.74 and 2.70 X 10(7) min-1 M-1 for K+1; 0.87 and 0.93 min-1 for k-1) but 20-40 times slower at 0-4 degrees C (0.13 and 0.096 X 10(7) min-1 M-1 for k+1; 0.031 and 0.022 min-1 for k-1 in cerebral cortical and cardiac membranes, respectively). Kinetic dissociation constants (Kds) were estimated to be 31.8 nM and 30.9 nM at 25 degrees C; 23.1 nM and 0-4 degrees C for the cerebral cortex and heart, respectively. In saturation studies, [3H]AF-DX 116 labeled 29 percent of the total [3H](-)QNB binding sites in the cerebral cortical membranes and 87 percent in the cardiac membranes, with Kd values of 28.9 nM and 17.9 nM, respectively. Muscarinic antagonists inhibited [3H]AF-DX 116 binding in a rank order of potency of atropine greater than dexetimide greater than AF-DX 116 greater than PZ greater than levetimide in both tissues. Except for PZ/[3H]AF-DX 116 and AF-DX 116/[3H]AF-DX 116 in the cerebral cortex, all the antagonist competition curves had Hill coefficients close to one. Carbachol and oxotremorine produced shallow inhibition curves against [3H]AF-DX 116 binding in both tissues. Regional distribution studies with [3H](-)QNB, [3H]PZ and [3H]AF-DX 116 showed that most of the muscarinic receptors in the cerebral cortex, hippocampus, nucleus accumbens and corpus striatum are of the M1 subtype while those in the brainstem, cerebellum and other lower brain regions are of the M2 subtype. These results indicate that [3H]AF-DX 116 is a useful probe for the study of heterogeneity of muscarinic cholinergic receptors.

Prodynorphin and vasopressin mRNA levels are differentially affected by chronic ethanol ingestion in the mouse

Opioid peptides derived from the precursor, prodynorphin, are co-localized with vasopressin in the hypothalamus and posterior pituitary, and vasopressin and prodynorphin synthesis are coordinately regulated during salt-loading. We had previously found that chronic ethanol ingestion resulted in decreased levels of hypothalamic and extrahypothalamic vasopressin mRNA, and the current study investigated the effect of ethanol ingestion on prodynorphin mRNA levels. A cRNA probe was constructed from a PCR product amplified from mouse genomic DNA. Cloning and sequencing of the PCR product revealed that the sequence of the mouse prodynorphin gene used to synthesize the probe is highly conserved, with high sequence similarity to corresponding regions of the gene in other mammalian species. In situ hybridization using the cRNA probe showed a widespread distribution of prodynorphin mRNA in mouse brain. In dehydrated mice, prodynorphin mRNA was significantly increased in the hypothalamus and nearly all other brain areas examined. In ethanol-fed mice, prodynorphin mRNA was also significantly increased in hypothalamus (50-60%) and in most brain areas. In the same mice, measurement of hypothalamic vasopressin mRNA confirmed a significant (approximately 60%) decrease. These results indicate that hypothalamic vasopressin and prodynorphin mRNA can be differentially regulated in certain situations.