Peter Kasa

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.

The cholinergic systems in brain and spinal cord.

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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.

Apolipoprotein D in the aging brain and in Alzheimer's dementia

Abstract Apolipoprotein D (apoD) levels were examined in the temporal cortex as well as an assessment of the location of apoD positive cells within the brain by immunohistochemical and biochemical methods in young control (YC), aged control (AC), and Alzheimer’s demented (AD) probands. Scattered apoD positive astrocytes and oligodendrocytes were found throughout the white matter by immunohistochemistry. ApoD immunoreactivity was also observed In the cerebellar oligodendrocytes of the YC group. There was faint positive apoD staining in scattered cortical astrocytes and a few neurons in the same group. In contrast, some of the AC and all of the AD probands had intense and frequent apoD immunostained cortical astrocytes and pyramidal neurons. The cortical senile plaques and neurofibrillary tangles were apoD immunonegative. No quantitative differences were found between the cortical apoD levels in the AC and AD groups, determined by immunoblotting. ApoD detected in the brain tissue was different in molecular weight (29 kDal) from that seen in CSF or in the serum (32 kDal). Our results indicate apoD is present in the human brain, especially in glial cells, and has increased abundance in the elderly and AD subjects [Neurol Res 2000; 22: 330-336]

Distribution of astroglia in glomeruli of the rat main olfactory bulb: Exclusion from the sensory subcompartment of neuropil

During an entire lifetime, sensory axons of regenerating olfactory receptor neurons can enter glomeruli in the olfactory bulb and establish synaptic junctions with central neurons. The role played by astrocytes in this unique permissiveness is still unclear. Glomerular astrocytes have been identified by immunocytochemistry for glial fibrillary acidic protein and S100 proteins at the light and electron microscopic levels. The latter labeling included submicroscopic lamellar and filopodial extensions of astroglial processes. Cell bodies and processes accumulate along the border between juxtaglomerular walls and glomerular neuropil. Within glomeruli, a network of astroglial processes encloses mesh‐like neuropil zones devoid of astroglia. Electron microscopy confirmed the division into subcompartments of glomerular neuropil: 1) The “sensory‐synaptic subcompartment” includes all sensory axon terminals and terminal dendritic branches receiving sensory input, whereas astroglia are excluded; 2) in the “central‐synaptic subcompartment,” astroglial processes are intermingled with other neuropil components: dendrites of relay cells and interneurons, dendrodendritic synapses, centrifugal (cholinergic and serotonergic) axons, their axodendritic synapses, and blood vessels. Unevenly distributed astroglial processes in this subcompartment are attached to vascular basal laminae, stem dendrites, and subpopulations of dendrodendritic synapses, especially those colocalized with centrifugal projections (“triadic synapses”). Astroglia‐free parts of the “central” subcompartment contain segments of dendrites and subpopulations of dendrodendritic synapses. Because of the subdivision of the glomerular neuropil into portions with and without glial components, glia do not completely demarcate the border between the “sensory” and the “central” subcompartments. Interdigitation between the subcompartments varies among glomeruli and even within a single glomerulus. The mesh width of astroglial networks covaries with numerical relations between sensory and dendrodendritic synapses. This distribution pattern of astrocytes suggests that these glial cells monitor brain‐derived effects on olfactory glomerular neuropil rather than olfactory input and that astroglial processes are (re‐)arranged accordingly. J. Comp. Neurol. 388:191–210, 1997.

Synaptic and non-synaptic cholinergic innervation of the various types of neurons in the main olfactory bulb of adult rat: Immunocytochemistry of choline acetyltransferase

The cholinergic neuronal structures and their synaptic connections in the main olfactory bulb of adult rats were analysed by using choline acetyltransferase immunocytochemistry. Within the glomeruli, cholinergic nerve fibers were restricted to strands which subdivided the neuropil into small compartments, the interior of which contained sensory axons but was devoid of cholinergic axons. Small numbers of choline acetyltransferase neurons were detected in all layers. Ultrastructural analysis revealed selective triadic synaptic relationships with different neuron classes in the intraglomerular area and in the external plexiform layer. These triads were made up of (i) a cholinergic axon, (ii) one or several periglomerular or granule cell dendrites, and (iii) usually one relay cell dendrite. In these triads, asymmetric cholinergic synapses were selectively focused on dendrites (gemmules and spines) of periglomerular or granule cells. Within the glomerulus, mitral and tufted cell dendrites were closely apposed to some cholinergic axon varicosities, most abundantly near arborizations of the apical dendrites. However, cholinergic synapses were never seen on any relay cell dendrite. In the external plexiform layer, cholinergic synapses were present on all parts of the superficial short-axon cells. In the internal plexiform layer and the granule cell layer, cholinergic axon varicosities exhibited close apposition or asymmetric synapses with granule cell gemmules. The data suggest that cholinergic projections from the basal forebrain to the main olfactory bulb focus synaptic innervation on interneurons. On relay cells, direct acetylcholine effects may occur, but these must be based on non-synaptic acetylcholine release at the surface of their dendrites.

Donepezil dose-dependently inhibits acetylcholinesterase activity in various areas and in the presynaptic cholinergic and the postsynaptic cholinoceptive enzyme-positive structures in the human and rat brain.

In the symptomatic treatment of mild to moderately severe dementia associated with Alzheimers disease, donepezil (E2020) has been introduced for the inhibition of acetylcholinesterase activity in the human brain. However, there is no morphological evidence as to how this chemical agent affects the acetylcholinesterase-positive structures in the various areas of the human and the rat CNS. This study demonstrates by histochemical means that donepezil exerts a dose-dependent inhibitory effect in vitro on acetylcholinesterase activity. The most sensitive areas were the cortex and the hippocampal formation. Within the different layers of the cortex, the cholinoceptive acetylcholinesterase-positive postsynaptic pyramidal cell bodies were more sensitive than the presynaptic cholinergic axonal processes. In the cortex, the cell body staining was already abolished by even 2 x 10(-8)M donepezil, whereas the axonal staining could be eliminated only by at least 5 x 10(-8)M donepezil. In the hippocampus, the axonal acetylcholinesterase reaction end-product was eliminated by 5 x 10(-7)M donepezil. The most resistant region was the putamen, where the staining intensity was moderately reduced by 1 x 10(-6)M donepezil. In the rat brain, the postsynaptic cholinoceptive and presynaptic cholinergic structures were inhibited by nearly the same dose of donepezil as in the human brain. These histochemical results provide the first morphological evidence that, under in vitro circumstances, donepezil is not a general acetylcholinesterase inhibitor in the CNS, but rather selectively affects the different brain areas and, within these, the cholinoceptive and cholinergic structures. The acetylcholinesterase staining in the nerve fibers (innervating the intracerebral blood vessels of the human brain and the extracerebral blood vessels of the rat brain) and at the neuromuscular junction in the diaphragm and gastrocnemius muscle of rat, was also inhibited dose dependently by donepezil. It is concluded that donepezil may be a valuable tool with which to influence both the pre- and the postsynaptic acetylcholinesterase-positive structures in the human and rat central and peripheral nervous systems.

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.

Reversible and irreversible acetylcholinesterase inhibitors cause changes in neuronal amyloid precursor protein processing and protein kinase C level in vitro.

The alternative routes of cleavage of the amyloid precursor protein (APP) result in the generation and secretion of both soluble APP and beta-amyloid, the latter being the main component of the amyloid deposits in the brains of individuals with Alzheimers disease (AD). This study examined the question of whether acetylcholinesterase (AChE) inhibitors can alter the processing of APP and the level of protein kinase C (PKC) in primary rat basal forebrain cultures. Western blotting was used to test two AChE inhibitors (reversible and irreversible) for their ability to enhance the release of APP and PKC content. These inhibitors were ambenonium (AMB) and metrifonate (MTF), at different concentrations. A significant increase was found in the cell-associated APP level in a basal forebrain neuronal culture, and there was an elevation of the APP release into the medium. Increases were similarly observed in the PKC levels after AMB or MTF treatment. The results suggest that these AChE inhibitors promote the non-amyloidogenic route of APP processing, which may be due to their stimulatory effects on PKC. The PKC activation may enhance the alpha-secretase activity and consequently the production of the N-terminal APP. Since both a decreased level of APP secretion and a low activity and level of PKC may be involved in the pathogenesis of AD, it is concluded that the administration of AChE inhibitors to AD patients may facilitate the memory processes and exert a neuroprotective effect.