Huperzine A Reverses Cholinergic and Monoaminergic Dysfunction Induced by Bilateral Nucleus Basalis Magnocellularis Injection of β-Amyloid Peptide (1–40) in Rats

(1) Huperzine A, a promising therapeutic agent for Alzheimer’s disease (AD), was tested for its effects on cholinergic and monoaminergic dysfunction induced by injecting β-amyloid peptide-(1–40) into nucleus basalis magnocellularis of the rat. (2) Bilateral injection of 10 μg β-amyloid peptide-(1–40) into nucleus basalis magnocellularis produced local deposits of amyloid plaque and functional abnormalities detected by microdialysis. In medial prefrontal cortex, reductions in the basal levels and stimulated release of acetylcholine, dopamine, norepinephrine, and 5-hydroxytryptamine were observed. However, oral huperzine A (0.18 mg/kg, once daily for 21 consecutive days) markedly reduced morphologic abnormalities at the injection site in rats infused with β-amyloid peptide-(1–40). Likewise, this treatment ameliorated the β-amyloid peptide-(1–40)-induced deficits in extracellular acetylcholine, dopamine, and norepinephrine (though not 5-hydroxytryptamine) in medial prefrontal cortex, and lessened the reduction in nicotine or methoctramine-stimulated release of acetylcholine and K⁺-evoked releases of acetylcholine and dopamine. (3) The present results provide the first direct evidence that huperzine A acts to oppose neurotoxic effects of β-amyloid peptide on cholinergic, dopaminergic, and noradrenergic systems of the rat forebrain.     

The β-Amyloid Precursor Protein (APP) and Alzheimer's Disease: Does the Tail Wag the Dog?

The β-amyloid precursor protein has been the focus of much attention from the Alzheimer's disease community for the past decade and a half. The β-amyloid precursor protein holds a pivotal position in Alzheimer's disease research because it is the precursor to the amyloid β-protein which many believe plays a central role in Alzheimer's disease pathogenesis. It was also the first gene in which mutations associated with inherited Alzheimer's disease were found. Although the molecular details of the generation of amyloid β-protein from β-amyloid precursor protein are being unraveled, the actual physiological functions of β-amyloid precursor protein are far from clear. This situation is changing as accumulating new evidence suggests that the C-terminal cytosolic tail of β-amyloid precursor protein may have multiple biological activities, ranging from axonal transport to nuclear signaling. This article reviews the current state of knowledge about the biological functions of β-amyloid precursor protein .     

S100β Increases Levels of β-Amyloid Precursor Protein and Its Encoding mRNA in Rat Neuronal Cultures

Abstract: S100β has been implicated in the formation of dystrophic neurites, overexpressing β-amyloid precursor protein (βAPP), in the β-amyloid plaques of Alzheimer's disease. We assessed the effects of S100β on cell viability of, neurite outgrowth from, and βAPP expression by neurons in primary cultures from fetal rat cortex. S100β (1–10 ng/ml) enhanced neuronal viability (as assessed by increased mitochondrial activity and decreased lactic acid dehydrogenase release) and promoted neurite outgrowth. Higher levels of S100β (100 ng/ml, but not 1 µg/ml) produced qualitatively similar, but less marked, effects. S100β also induced increased neuronal expression of the microtubule-associated protein MAP2, an effect that is consistent with trophic effects of S100β on neurite outgrowth. S100β (10 and 100 ng/ml) induced graded increases in neuronal expression of βAPP and of βAPP mRNA. These results support our previous suggestion that excessive expression of S100β by activated, plaque-associated astrocytes in Alzheimer's disease contributes to the appearance of dystrophic neurites overexpressing βAPP in diffuse amyloid deposits, and thus to the conversion of these deposits into the diagnostic neuritic β-amyloid plaques.     

Identification of a Chymotrypsin-Like Mast Cell Protease in Rat Brain Capable of Generating the N-Terminus of the Alzheimer Amyloid β-Protein

Abstract: Cleavage after Met⁵⁹⁶ of the β-amyloid precursor protein to generate the N-terminus of β-protein indicates the activity of a protease having chymotrypsin-like specificity. A chymotrypsin-like protease is further implicated in Alzheimer's disease by the increased synthesis of the protease inhibitor α₁-antichymotrypsin in pathologically affected brain regions and by the presence in the amyloid deposits of inactivated forms of α₁-antichymotrypsin (indicating irreversible binding to a target chymotrypsin-like protease). In the present report, we have purified from rat brain a chymotrypsin-like protease that (a) binds with high affinity to human α₁-antichymotrypsin, (b) proteolytically generates a β-protein-containing C-terminal fragment from full-length recombinant human β-amyloid precursor protein, and (c) selectively cleaves methoxysuccinyl-Glu-Val-Lys-Met-p-nitroanilide (a substrate modeling the protease recognition domain for the β-protein N-terminal cleavage site). Amino acid sequences of tryptic fragments of the purified rat brain chymotrypsin-like protease indicate an identity with rat mast cell protease I. Moreover, the ontogeny and compartmentalization of rat brain chymotrypsin-like protease are consistent with those of connective tissue-type mast cells in the meningeal and intracortical perivasculature. Because these areas in human brain form extensive β-amyloid deposits in Alzheimer's disease, Down's syndrome, and hereditary cerebral hemorrhage with amyloidosis of Dutch origin, the present findings suggest that a brain mast cell chymotrypsin-like protease may participate in generating perivascular β-protein, which ultimately aggregates into β-amyloid deposits.     

Structure-Activity Analyses of β-Amyloid Peptides: Contributions of the β25–35 Region to Aggregation and Neurotoxicity

Abstract: The neurodegeneration of Alzheimer's disease has been theorized to be mediated, at least in part, by insoluble aggregates of β-amyloid protein that are widely distributed in the form of plaques throughout brain regions affected by the disease. Previous studies by our laboratory and others have demonstrated that the neurotoxicity of β-amyloid in vitro is dependent upon its spontaneous adoption of an aggregated structure. In this study, we report extensive structure-activity analyses of a series of peptides derived from both the proposed active fragment of β-amyloid, β25–35, and the full-length protein, β1–42. We examine the effects of amino acid residue deletions and substitutions on the ability of β-amyloid peptides to both form sedimentable aggregates and induce toxicity in cultured hippocampal neurons. We observe that significant levels of peptide aggregation are always associated with significant β-amyloid-induced neurotoxicity. Further, both N- and C-terminal regions of β25–35 appear to contribute to these processes. In particular, significant disruption of peptide aggregation and toxicity result from alterations in the β33–35 region. In β1–42 peptides, aggregation disruption is evidenced by changes in both electrophoresis profiles and fibril morphology visualized at the light and electron microscope levels. Using circular dichroism analysis in a subset of peptides, we observed classic features of β-sheet secondary structure in aggregating, toxic β-amyloid peptides but not in nonaggregating, nontoxic β-amyloid peptides. Together, these data further define the primary and secondary structures of β-amyloid that are involved in its in vitro assembly into neurotoxic peptide aggregates and may underlie both its pathological deposition and subsequent degenerative effects in Alzheimer's disease.     

Change of cholinergic transmission and memory deficiency induced by injection of β-amyloid protein into NBM of rats

The change of cholinergic transmission of ?-amyloid protein (β-AP) treated rats was studied by intracerebral microdialysis sampling combined with HPLC analysis. β-AP_1—40 was injected into nucleus basalis magnocellularis (NBM). Passive avoidance response test (step-down test) and delayed alternation task were used for memory testing. The impairment of memory after injection of β-AP_1—40 into NBM exhibited mainly the deficiency of short-term working memory. One week after injection of β-AP_1—40 the release of acetylcholine (ACh) from frontal cortex of freely-moving rats decreased significantly, and the response of cholinergic nerve ending to the action of high [K⁺] solution was rather weak. In control animals the percentage of increase of ACh-release during behavioral performance was 57%, while in β-AP_1—40-treated rats it was 34%. The temporary increase of the ACh-release of the rat put into a new place was also significantly diminished in β-AP_1—40-treated rats. The results show that the injection of β-AP_1—40 into NBM impairs the cholinergic transmission in frontal cortex, and the impairment of cholinergic transmission may be the main cause of the deficit of working memory.     

Change of cholinergic transmission and memory deficiency induced by injection of β-amyloid protein into NBM of rats

The change of cholinergic transmission of β-amyloid protein (β-AP) treated rats was studied by intracerebral microdialysis sampling combined with HPLC analysis. β-AP_1-40 was injected into nucleus basalis magnocellularis (NBM). Passive avoidance response test (step-down test) and delayed alternation task were used for memory testing. The impairment of memory after injection of β-AP_1-40 into NBM exhibited mainly the deficiency of short-term working memory. One week after injection of β-AP^1-40 the release of acetylcholine (ACh) from frontal cortex of freely-moving rats decreased significantly, and the response of cholinergic nerve ending to the action of high [K⁺] solution was rather weak. In control animals the percentage of increase of AChrelease during behavioral performance was 57%, while in β-AP^1-40-treated rats it was 34%. The temporary increase of the ACh-release of the rat put into a new place was also significantly diminished in β-AP_1-40 -treated rats. The results show that the injection of β-AP_1-40 into NBM impairs the cholinergic transmission in frontal cortex, and the impairment of cholinergic transmission may be the main cause of the deficit of working memory.     

Aggregation of β-Amyloid Peptide Is Promoted by Membrane Phospholipid Metabolites Elevated in Alzheimer's Disease Brain

Abstract: Increased amounts of β-amyloid (Aβ) peptide deposits are found in Alzheimer's disease brain. These amyloid deposits have been implicated in the pathophysiology of this common dementing illness. Aβ peptides have been shown to be toxic to neurons in cell culture, and this toxicity is critically dependent on the aggregation of the peptide into cross-β-pleated sheet fibrils. Also, in vivo and postmortem NMR studies have shown changes in certain brain membrane phospholipid metabolites in normal aging and more extensive alterations in patients with Alzheimer's disease. The finding that membrane phospholipids affect the aggregation of Aβ suggests that the abnormalities in membrane metabolism found in Alzheimer's disease could affect the deposition of Aβ in vivo. Therefore, we examined the effect of membrane phospholipid metabolites that are altered in Alzheimer's disease brain on the aggregation of Aβ(1–40) using a light scattering method. Certain metabolites (glycerophosphocholine, glycerophosphoethanolamine, and α-glycerophosphate) augment the aggregation of Aβ. Other membrane phospholipid metabolites (phosphocholine, phosphoethanolamine, and inositol-1-phosphate) have no effect. We conclude that increased membrane phospholipid metabolite concentrations may play a role in the deposition of Aβ seen in normal aging and the even greater deposition of Aβ observed in Alzheimer's disease.     

Differences Between Vascular and Plaque Core Amyloid in Alzheimer's Disease

Abstract: The predominant protein of cerebrovascular and plaque core amyloid in Alzheimer's disease, Down's syndrome, hereditary hemorrhage with amyloidosis—Dutch type, sporadic cerebral amyloid angiopathy, and age-related amyloidosis is a unique polypeptide, called β protein. The length of the plaque amyloid protein was reported to be 42–43 residues, but the complete length of the cerebral vascular amyloid is not known. To clarify this issue, amyloid fibrils from the leptomeninges of an Alzheimer's disease patient were isolated and the primary structure determined. The complete sequence of cerebrovascular β-amyloid protein, although homologous to the plaque core amyloid protein previously reported, has 39 residues instead of 42. Amino terminal heterogeneity is present but minimal, and it is three residues shorter at the carboxy terminus. These differences are similar to those found in two cases of hereditary hemorrhage with amyloidosis—Dutch type. The differences between vascular and plaque β-amyloid may reflect diverse processing of the β protein precursor in the vessel wall and brain parenchyma due to tissue-specific endopeptidases.     

<Emphasis Type="Italic">In silico</Emphasis> modeling of the specific inhibitory potential of thiophene-2,3-dihydro-1,5-benzothiazepine against BChE in the formation of β-amyloid plaques associated with Alzheimer's disease

Background  

Alzheimer's disease, known to be associated with the gradual loss of memory, is characterized by low concentration of acetylcholine in the hippocampus and cortex part of the brain. Inhibition of acetylcholinesterase has successfully been used as a drug target to treat Alzheimer's disease but drug resistance shown by butyrylcholinesterase remains a matter of concern in treating Alzheimer's disease. Apart from the many other reasons for Alzheimer's disease, its association with the genesis of fibrils by β-amyloid plaques is closely related to the increased activity of butyrylcholinesterase. Although few data are available on the inhibition of butyrylcholinesterase, studies have shown that that butyrylcholinesterase is a genetically validated drug target and its selective inhibition reduces the formation of β-amyloid plaques.     

Oxidative Stress Induces Dephosphorylation of τ in Rat Brain Primary Neuronal Cultures

Abstract: Oxidative stress and free radical damage have been implicated in the neurodegenerative changes characteristic of several neurodegenerative diseases, including Alzheimer's disease. There is experimental evidence that the neurotoxicity of β-amyloid is mediated via free radicals, and as the deposition of β-amyloid apparently precedes the formation of paired helical filaments (PHF) in Alzheimer's disease, we have investigated whether subjecting primary neuronal cultures to oxidative stress induces changes in the phosphorylation state of the principal PHF protein τ that resemble those found in PHF-τ. Contrary to causing an increase in τ phosphorylation, treatment of neurones with hydrogen peroxide caused a dephosphorylation of τ and so we conclude that oxidative stress is not the direct cause of τ hyperphosphorylation and hence of PHF formation.     

β-Amyloid Neurotoxicity in Human Cortical Culture Is Not Mediated by Excitotoxins

Abstract: β-Amyloid is a metabolic product of the amyloid precursor protein, which accumulates abnormally in senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of 0-amyloid has been observed in cell culture and in vivo, but the mechanism of this effect is unclear. In this report, we describe the direct neurotoxicity of β-amyloid in high-density primary cultures of human fetal cortex. In 36-day-old cortical cultures, β-amyloid neurotoxicity was not inhibited by the broad-spectrum excitatory amino acid receptor antagonist kynurenate or the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid under conditions that inhibited glutamate and NMDA neurotoxicity. In 8-day-old cortical cultures, neurons were resistant to glutamate and NMDA toxicity but were still susceptible to β-amyloid neurotoxicity, which was unaffected by excitatory amino acid receptor antagonists. Treatment with β-amyloid caused chronic neurodegenera-tive changes, including neuronal clumping and dystrophic neurites, whereas glutamate treatment caused rapid neuronal swelling and neurite fragmentation. These results suggest that β-amyloid is directly neurotoxic to primary human cortical neurons by a mechanism that does not involve excitatory amino acid receptors.     

Role of A β and the α 7 nicotinic acetylcholine receptor in regulating synaptic plasticity in Alzheimer's disease

Alzheimer's disease (AD) is caused by the accumulation of β-amyloid protein (Aβ) in the brain. The aggregation of β-amyloid protein to higher molecular weight fibrillar forms is also considered to be an important step in the pathogenesis of the disease. The memory problems associated with AD are likely to be caused by changes in synaptic plasticity. Recent studies suggest that Aβ binds to the α 7 nicotinic acetylcholine receptor (α 7 nAChR), which plays an important role in synaptic plasticity and memory. A loop domain localized towards the C-terminus of the extracellular region of the receptor has been identified as forming part of a putative Aβ-binding site. In cell culture experiments, the binding of Aβ to the α 7 nAChR has been found to cause an increase in the level of acetylcholinesterase, which is also increased around amyloid plaques in the AD brain. These studies indicate that the Aβ-binding site on the α 7 nAChR receptor is an important new target for therapeutic development in AD.     

Beta-secretase-cleaved amyloid precursor protein in Alzheimer brain: a morphologic study

β-amyloid (Aβ) is the main constituent of senile plaques seen in Alzheimer's disease. Aβ is derived from the amyloid precursor protein (APP) via proteolytic cleavage by proteases β- and β-secretase. In this study, we examined content and localization of β-secretase-cleaved APP (β-sAPP) in brain tissue sections from the frontal, temporal and occipital lobe. Strong granular β-sAPP staining was found throughout the gray matter of all three areas, while white matter staining was considerably weaker. β-sAPP was found to be localized in astrocytes and in axons. We found the β-sAPP immunostaining to be stronger and more extensive in gray matter in Alzheimer disease (AD) cases than controls. The axonal β-sAPP staining was patchy and unevenly distributed for the AD cases, indicating impaired axonal transport. β-sAPP was also found surrounding senile plaques and cerebral blood vessels. The results presented here show altered β-sAPP staining in the AD brain, suggestive of abnormal processing and transport of APP.     

Choline administration: central effect mediated by stimulation of acetylcholine synthesis.

Oral administration of either choline or physostigmine to rats stimulated the metabolism of dopamine in brain. Treatment of rats with an acetylcholine receptor blocker (atropine) or an inhibitor of acetylcholine synthesis [4-(1-naphthylvinyl) pyridine] antagonized the choline-induced increase in dopamine metabolism. In contrast to its effect on the choline-induced increase in dopamine metabolism, 4-(1-naphthylvinyl) pryridine did not prevent the increase induced by physostigmine. These results indicate that oral administration of choline augments central cholinergic function, and suggest that the mechanism involves stimulation of the rate of synthesis and release of acetylcholine.     

Basal Forebrain Cholinergic Dysfunction in Alzheimer’s Disease – Interrelationship with β-amyloid, Inflammation and Neurotrophin Signaling

Alzheimer’s disease, the most common neurodegenerative disorder of senile dementia, is characterized by two major morpho-pathological hallmarks. Deposition of extracellular neuritic, β-amyloid peptide-containing plaques (senile plaques) in cerebral cortical regions of Alzheimer patients is accompanied by the presence of intracellular neurofibrillary tangles in cerebral pyramidal neurons. Basal forebrain cholinergic dysfunction is also a consistent feature of Alzheimer’s disease, which has been suggested to cause, at least partly, the cognitive deficits observed in patients with Alzheimer’s disease. Impaired cortical cholinergic neurotransmission may also contribute to β-amyloid plaque pathology in Alzheimer’s disease by affecting expression and processing of the β-amyloid precursor protein (APP). Vice versa, low level of soluble β-amyloid has been observed to inhibit cholinergic synaptic function. Deposition of β-amyloid plaques in Alzheimer’s disease is also accompanied by a significant plaque-associated glial up-regulation of interleukin-1, which has been attributed to affect expression and metabolism of APP and to interfere with cholinergic transmission. Understanding the molecular mechanisms underlying the interrelationship between cortical cholinergic dysfunction, β-amyloid formation and deposition, as well as local inflammatory upregulation, would allow to derive potential treatment strategies to pharmacologically intervene in the disease-causing signaling cascade.     

Accumulation of spectrin and calpain-cleaved spectrin breakdown products in CSF after traumatic brain injury in rats

The introduction of acetylcholine esterase inhibitors for symptomatic treatment of Alzheimer's disease, and the promise of drugs that may delay disease progression, has created a great need for reliable diagnostic tools. However, current criteria for the clinical diagnosis of AD are largely based on the exclusion of other dementia disorders and disease markers are lacking. Since biochemical changes in the brain are reflected in the cerebrospinal fluid (CSF), the search for diagnostic tools for AD has been directed toward CSF markers. CSF markers for AD should reflect the central pathogenic processes of the disorder, i.e. the mismetabolism of β-amyloid (Aβ) and the hyperphosphorylation of tau. Several studies have found that the CSF level of Aβ42 is decreased, and the CSF levels of total tau and phosphorylated tau are increased in AD as compared with normal controls. Thus, the sensitivity of these changes in AD is high. But changes in CSF-Ab42 and CSF-tau have been found in other neurodegenerative disorders and therefore, the specificity seems to be moderately high. Other potential markers that may increase the clinical diagnostic accuracy include the CSF/serum albumin ratio (for identification of blood–brain barrier damage related to disturbances in the small intracerebral vessels), CSF-sulfatide (for identification of ongoing demyelination related to white matter changes and CSF-neurofilament light protein (NFL) [for identification of ongoing axonal (tau and NFL) degeneration]. Use of the summarized information from analyses of several CSF biochemical markers, from the clinical examination, and from brain imaging (SPECT, CT/MRI) may increase the accuracy of the clinical diagnosis.     

Modeling Alzheimer's disease and other proteopathies in vivo: is seeding the key?

Summary.  Protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease, triplet repeat disorders, tauopathies, and prion diseases. In many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. Possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide range of diseases. However, testing compounds preclinically will require disease-relevant animal models. Numerous transgenic mouse models of β-amyloidosis, tauopathy, and other aspects of AD have now been produced, but none of the existing models fully recapitulates the pathology of AD. In an attempt to more faithfully replicate the human disease, we infused dilute AD-brain extracts into Tg2576 mice at 3-months of age (i.e. 5–6 months prior to the usual onset of β-amyloid deposition). We found that intracerebral infusion of AD brain extracts results in: 1) Premature deposition of β-amyloid in eight month-old, β-amyloid precursor protein (βAPP)-transgenic mice (Kane et al., 2000); 2) augmented amyloid load in the injected hemisphere of 15 month-old transgenic mice; 3) evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and 4) tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. The seeding of β-amyloid in vivo by AD brain extracts suggests pathogenic similarities between β-amyloidoses such as AD and other cerebral proteopathies such as the prionoses, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases. Received June 28, 2001 Accepted August 6, 2001 Published online June 26, 2002     

Alterations of Acetylcholine and Choline Metabolism in Mammalian Preparations Treated with β-Bungarotoxin

Abstract: We have studied the effects of β-bungarotoxin on acetylcholine and choline metabolism in central and peripheral cholinergic preparations using a gas chromatographic-mass spectrometric assay for acetylcholine and choline. In contrast with previous reports, β-bungarotoxin did not inhibit the high-affinity uptake of labeled choline or the synthesis of acetylcholine in rat brain synaptosomal fractions. However, the toxin did cause a significant increase of medium choline when it was incubated with synaptosomal fractions. This increase of endogenous choline in the medium may account for the previously reported inhibition of choline uptake because of a dilution of the specific activity of the labeled choline in the medium. Several experiments are reported in which a further characterization was made of the effect of β-bungarotoxin on medium choline. β-Bungarotoxin was also shown to cause a large increase of acetylcholine release from rat brain minces and a depletion of the acetylcholine content of minces. A similar phenomenon was found in diaphragm preparations that were exposed continuously to β-bungarotoxin. However, diaphragms that were treated for only 30 min with toxin showed the previously reported increase of acetylcholine content. β-Bungarotoxin did not have any measurable effect on acetylcholine turnover in smooth muscle preparations from guinea pig ileum. These results help to explain certain inconsistencies in the literature regarding the action of β-bungarotoxin.     

Acetylcholine turnover rates in rat brain regions during cocaine self-administration

The involvement of cholinergic neurons in the brain processes underlying reinforcement has been recently demonstrated. This experiment assessed the potential role of cholinergic neurons in cocaine reinforcement by measuring the turnover rates of acetylcholine in brain regions of rats self-administering cocaine and in yoked cocaine and yoked vehicle-infused controls. The activity of cholinergic innervations of and/or interneurons in the olfactory tubercle, caudate putamen, diagonal band-pre-optic region, ventral pallidum, lateral and medial hypothalamus, hippocampus, ventral tegmental area and visual cortices reflected by the turnover rates of acetylcholine were significantly altered in rats self-administering cocaine compared to yoked cocaine infused controls. These changes implicate the involvement of cholinergic neurons with cell bodies in the diagonal band-pre-optic region, the medial septum and several brainstem nuclei and interneurons in the caudate-putamen and ventral pallidum in the processes underlying cocaine self-administration. The identified cholinergic neuronal systems may have a broader role in the brain processes for natural reinforcers (i.e. food, water, etc.) since drugs of abuse are believed to produce reinforcing effects through these systems.