Irreversible dimerization/tetramerization and post-translational modifications inhibit proteolytic degradation of A beta peptides of Alzheimer's disease.

Experimental evidence increasingly implicates the beta-amyloid peptide in the pathogenesis of Alzheimer's disease. Beta-amyloid filaments dramatically accumulate in the neuritic plaques and vascular deposits as the result of the brain's inability to clear these structures. In this paper, we demonstrate that in addition to the intrinsic stability of A beta N-42, the time dependent generation of irreversibly associated A beta dimers and tetramers incorporated into A beta filaments are themselves resistant to proteolytic degradation. The presence of post-translational modifications such as isomerization of aspartyls 1 and 7, cyclization of glutamyl 3 to pyroglutamyl and oxidation of methionyl 35, further contribute to the insolubility and stability of A beta. All these factors promote the accumulation of neurotoxic amyloid in the brains of patients with Alzheimer's disease, and should be considered in therapeutic strategies directed towards the dissociation of the brain's A beta filaments.

The role of complement in Alzheimer's disease pathology.

Complement proteins are integral components of amyloid plaques and cerebral vascular amyloid in Alzheimer brains. They can be found at the earliest stages of amyloid deposition and their activation coincides with the clinical expression of Alzheimer's dementia. This review will examine the origins of complement in the brain and the role of beta-amyloid peptide (Abeta) in complement activation in Alzheimer's disease, an event that might serve as a nidus of chronic inflammation. Pharmacology therapies that may serve to inhibit Abeta-mediated complement activation will also be discussed.

Oligomerizaiton and fibril asssembly of the amyloid-beta protein.

In this chapter, we attempt to analyze the evolution of the amyloid-beta (Abeta) molecular structure from its inception as part of the Abeta precursor protein to its release by the secretases and its extrusion from membrane into an aqueous environment. Biophysical studies suggest that the Abeta peptide sustains a series of transitions from a molecule rich in alpha-helix to a molecule in which beta-strands prevail. It is proposed that initially the extended C-termini of two opposing Abeta dimers form an antiparallel beta-sheet and that the subsequent addition of dimers generates a helical Abeta protofilament. Two or more protofilaments create a strand in which the hydrophobic core of the beta-sheets is shielded from the aqueous environment by the N-terminal polar domains of the Abeta dimers. Once the nucleation has occurred, the Abeta filament grows in length by the addition of dimers or tetramers.

The cholinergic deficit coincides with Abeta deposition at the earliest histopathologic stages of Alzheimer disease.

Effective therapeutic intervention in Alzheimer disease (AD) will be most effective if it is directed at early events in the pathogenic sequence. The cholinergic deficit may be such an early event. In the present study, the brains of 26 subjects who had no history of cognitive loss and who were in early histopathologic stages of AD (average Braak stage less than II) were examined at autopsy to determine whether a cortical cholinergic decrement was associated with Abeta concentration or deposition. In the superior frontal and inferior temporal gyri, the choline acetyltransferase (ChAT) activity of plaque-containing cases was significantly decreased (p < 0.05, unpaired, two-tailed t-tests), measuring 70.9% and 79.5%, respectively, relative to plaque-free cases. In the inferior temporal gyrus, Spearman's rank correlation analysis showed that ChAT activity had a significant inverse correlation with Abeta concentration (p = 0.075; r = -0.3552). The results indicate that the cholinergic deficit is established at an early histopathologic stage of AD, before the onset of clinical symptoms.

Apolipoprotein E alters metabolism of AbetaPP in cells engaged in beta-amyloidosis.

Canine smooth muscle cells (SMCs), cultured from amyloid-affected brain blood vessels accumulate Alzheimer amyloid-beta peptide (Abeta) intracellularly, either spontaneously or after treatment with apolipoprotein E (apoE). ApoE is codeposited with Abeta, which suggests that apoE participates in Abeta accumulation. We tested the hypothesis that apoE-induced accumulation of Abeta in SMCs is caused by an increased production of amyloid-beta precursor protein (AbetaPP) and/or its altered metabolism. We found that 24 hours of treatment with apoE3 or apoE4 induced intracellular accumulation of Abeta-immunoreactive deposits in SMCs but did not influence AbetaPP production and processing. The treatment with apoE3 or E4 for 3 days resulted in the following: increased Abeta-accumulation; reduced levels of secreted Abeta; increased production and cellular retention of mature AbetaPP770; and reduced culture growth, cell proliferation, and viability. ApoE4, but not apoE3, increased cellular levels of mRNA AbetaPP 770 (the main form produced in SMCs) about ninefold. ApoE3 stimulated production and cellular retention of endogenous apoE. We hypothesize that Abeta accumulation is triggered by apoE, which may bind and immobilize soluble Abeta produced in SMCs. The newly formed Abeta deposits may further accelerate Abeta accumulation by altering metabolism of AbetaPP.

PD 142676 (CI 1002), a novel anticholinesterase and muscarinic antagonist.

Inhibition of brain acetylcholinesterase (AChE) can provide relief from the cognitive loss associated with Alzheimer's disease (AD). However, unwanted peripheral side effects often limit the usefulness of the available anticholinesterases. Recently, we identified a dihydroquinazoline compound, PD 142676 (CI 1002) that is a potent anticholinesterase and a functional muscarinic antagonist at higher concentrations. Peripherally, PD 142676, unlike other anticholinesterases, inhibits gastrointestinal motility in rats, an effect consistent with its muscarinic antagonist properties. Centrally, the compound acts as a cholinomimetic. In rats, PD 142676 decreases core body temperature. It also increases neocortical arousal, as measured by quantitative electroencephalography, and cortical acetylcholine levels, measured by in vivo microdialysis. The compound improves the performance of C57/B10j mice in a water maze task and of aged rhesus monkeys in a delayed match-to-sample task involving short-term memory. The combined effect of AChE inhibition and muscarinic antagonism distinguishes PD 142676 from other anticholinesterases, and may be useful in treating the cognitive dysfunction of AD and produce fewer peripheral side effects.

Interleukin-1 beta dissociates beta-amyloid precursor protein and beta-amyloid peptide secretion.

A heightened production of interleukin 1 beta (IL-1 beta) has been reported in microglial-associated amyloid deposits in Alzheimer's disease (AD) brains. These plaques are composed predominantly of beta/A4 peptide derived from beta-amyloid precursor protein (beta APP). We demonstrate that short-term (1 h) IL-1 beta-treatment increases beta APPs secretion and concomitantly decreases cell-associated beta APP in human H4 neuroglioma cells. Long-term (5 h) IL-1 beta treatment did not alter secreted or cell-associated beta APP content. In contrast, the secretion of beta/A4-containing epitope was not affected by short-term IL-1 beta stimulation; however, long-term IL-1 beta treatment decreased the amount of beta/A4-containing epitope secreted from the cells. These results show that IL-1 beta modifies the processing and secretion of beta APP to exacerbate perhaps the neuropathology of AD.

Identification of a 3-hydroxylated tacrine metabolite in rat and man: metabolic profiling implications and pharmacology.

Discrepancies in urinary metabolic profiles in rats administered tacrine (1) suggested the presence of an unidentified metabolite of 1. Chromatographic methods were developed that allowed isolation of a metabolite fraction containing both 1-hydroxytacrine (2) and an unknown metabolite from rat urine. Mass spectral analysis indicated this metabolite to be a monohydroxylated derivative, which upon two dimensional COSY NMR analysis could be assigned as 3-hydroxytacrine (4). This structural assignment was confirmed by independent synthesis of 4. Compound 4 was also identified as a human urinary metabolite of 1. Biologically, 4 was found to have in vitro human red blood cell acetylcholinesterase inhibitory activity similar to that of 2 and 4-hydroxytacrine (5) and approximately 8-fold less than that of 1. These results underscore the need to conduct rigorous structural identification studies, especially in cases where isomeric metabolites are possible, in assessing the accuracy of chromatographic profiling techniques.

2-amino-4H-3,1-benzoxazin-4-ones as inhibitors of C1r serine protease.

A series of 2-amino-4H-3,1-benzoxazin-4-ones have been synthesized and evaluated as inhibitors of the complement enzyme C1r. C1r is a serine protease at the beginning of the complement cascade, and complement activation by beta-amyloid may represent a major contributing pathway to the neuropathology of Alzheimer's disease. Compounds such as 7-chloro-2-[(2-iodophenyl)-amino]benz[d][1,3]oxazin-4-one (32) and 7-methyl-2-[(2-iodophenyl)amino]benz[d][1,3]oxazin-4-one (37) show improved potency compared to the reference compound FUT-175. Many of these active compounds also possess increased selectivity for C1r compared to trypsin and enhanced hydrolytic stability relative to 2-(2-iodophenyl)-4H-3,1-benzoxazin-4-one (1).

Therapeutic intervention in dementia.

The search for novel therapeutics for human cognitive disorders has intensified. Neurotransmitter replacement therapies represent a short-term hope for treating cognitive dysfunction associated with Alzheimer's disease (AD). AD, however, is clearly a neurodegenerative disease and is characterized by a loss of synaptic elements. Ultimately, synaptic loss must be halted to alter the disease course. Agents mimicking or modulating the actions of neurotrophic factors may be useful. They may restore lost function and exert anabolic effects on existing neurons, making treated cells less susceptible to neurotoxic insult (i.e., excitotoxicity, oxidative stress, etc.). Intervening in the biogenesis of amyloid plaques and blunting local inflammatory responses may provide the ultimate treatment for AD. The success of any treatment, however, rests on early diagnosis. Early intervention in the neurodegenerative disease process will be required. Without early intervention, the risk of maintaining patients in a premorbid state is high. Therefore, it is likely that no single approach will provide optimal therapy for the AD patient and multifactorial treatment strategies may be required.

The role of arachidonic acid in the secretion of the amyloid precursor protein (APP).

We have studied the activation of human ml-muscarinic receptors in a genetically engineered Chinese hamster ovary cell line (CHO-ml) to determine which second messenger systems affect the secretion of APP via the non-amyloidogenic route. Carbachol activation of the signaling pathways in CHO-ml cells promotes APP secretion by activation of both protein kinase C (PKC)-dependent or Ca(++)-dependent second messenger pathways. Both pathways converge to increase the enzyme activity of phospholipase A2 (PLA2), the enzyme that releases arachidonic acid from cellular stores. Directly activating PLA2 with melittin, a peptide from bee venom, or by adding arachidonic acid directly to cultured cells increases the secretion of APP. Thus, our results indicate that arachidonic acid is yet another cellular second messenger involved in regulating the metabolism of APP in addition to PKC and cytoplasmic Ca++. Moreover, activation of PLA2 appears to be an obligatory event in increasing the secretion of APP from CHO-ml cells by the various methods of activation that we have tried thus far.

Traumatic brain injury elevates the Alzheimer's amyloid peptide A beta 42 in human CSF. A possible role for nerve cell injury.

The increased risk for Alzheimer's Disease (AD) associated with traumatic brain injury (TBI) suggests that environmental insults may influence the development of this age-related dementia. Recently, we have shown that the levels of the beta-amyloid peptide (A beta 1-42) increase in the cerebrospinal fluid (CSF) of patients after severe brain injury and remain elevated for some time after the initial event. The relationships of elevated A beta with markers of blood-brain barrier (BBB) disruption, inflammation, and nerve cell or axonal injury were evaluated in CSF samples taken daily from TBI patients. This analysis reveals that the rise in A beta 1-42 is best correlated with possible markers of neuronal or axonal injury, the cytoskeletal protein tau, neuron-specific enolase (NSE), and apolipoprotein E (ApoE). Similar or better correlations were observed between A beta 1-40 and the three aforementioned markers. These results imply that the degree of brain injury may play a decisive role in determining the levels of A beta 1-42 and A beta 1-40 in the CSF of TBI patients. Inflammation and alterations in BBB may play lesser, but nonetheless significant, roles in determining the A beta level in CSF after brain injury.

Cortical cholinergic denervation elicits vascular A beta deposition.

Selective destruction of the cholinergic nucleus basalis magnocellularis (nbm) in the rabbit by the p75 neurotrophin receptor (NTR) immunoglobulin G (IgG) complexed to the toxin saporin leads to the deposition of amyloid-beta (A beta) in and around cerebral blood vessels. In some instances, the perivascular A beta resemble the diffuse deposits observed in Alzheimer's disease (AD). We propose that cortical cholinergic deprivation results, among other perturbations, in the loss of vasodilation mediated by acetylcholine. In addition to a dysfunctional cerebral blood flow, alterations in vascular chemistry affecting endothelial and smooth muscle cells may result in cerebral hypoperfusion and a breached blood-brain barrier (BBB). The selective removal of the rabbit nbm and A beta accumulation may serve as an important nontransgenic, and more physiological, model for the testing of pharmacological and immunological agents designed to control the deposition and the deleterious effects of A beta in AD.

Beta-amyloid levels predict cholinesterase activity in human cerebrospinal fluid.

There is increasing evidence suggesting that beta-amyloid (Abeta) has a direct influence on cholinergic activity. In particular, Abeta has been shown to induce the expression of acetylcholinesterase in the brains of CT-100-expressing transgenic mice and in cell culture experiments. These data indicate a link exists between Abeta production and acetylcholinesterase expression in the human CNS. To test this hypothesis, Abeta levels and cholinesterase activity were measured in 110 human CSF samples. Abeta levels were found to have a significant and positive correlation with cholinesterase activity. This correlation was particularly strong in individuals > 50 years of age. These data support the hypothesis that Abeta can effect cholinergic activity and that this effect may be enhanced in the elderly.

Secretion and accumulation of Alzheimer's beta-protein by cultured vascular smooth muscle cells from old and young dogs.

Cultured smooth muscle cells isolated from beta-amyloid-affected blood vessels from old dogs accumulate beta-protein at early passages [5,24]. Now, we show that smooth muscle cells derived from amyloid-free brain blood vessels and peripheral arteries from old and young animals are induced by culture conditions to deposit intracellularly fibrillar and non-fibrillar beta-protein. Accumulation of beta-protein is associated with a higher secretion of beta-protein, but not with a higher secretion of beta-amyloid precursor protein (beta APP) or higher cellular content of beta APP. Gradual cessation of proliferative activity was observed in cultures that accumulate beta-protein.

Cholinergic deafferentation of the rabbit cortex: a new animal model of Abeta deposition.

Brain deposition of the amyloid beta-peptide (Abeta) is a critical step in the pathogenesis of Alzheimer's disease (AD) and human cerebral amyloid angiopathy (CAA). A small fraction of AD and CAA cases are caused by gene mutations leading to increased production and deposition of Abeta, but for the majority, there is no known direct genetic cause. We have hypothesized that Abeta deposition in these sporadic cases occurs as a result of cortical cholinergic deafferentation. Here we show that cortical cholinergic deafferentation, induced in rabbits by a selective immunotoxin, leads to Abeta deposition in cerebral blood vessels and perivascular neuropil. Biochemical measurements confirmed that lesioned animals had 2.5- and 8-fold elevations of cortical Abeta40 and Abeta42, respectively. Cholinergic deafferentation may be one factor that can contribute to Abeta deposition.

Hippocampal nitric oxide upregulation precedes memory loss and A beta 1-40 accumulation after chronic brain hypoperfusion in rats.

Chronic brain hypoperfusion (CBH) using permanent occlusion of both common carotid arteries in an aging rat model, has been shown to mimic human mild cognitive impairment (MCI), an acknowledged high risk condition that often converts to Alzheimer's disease. An aging rat model was used to determine whether hippocampal nitric oxide (NO) is abnormally expressed following CBH for two or eight weeks. At each time point, spatial memory was measured with the Morris water maze and hippocampal A beta 1-40/1-42 concentrations were obtained using sandwich ELISA. Real-time amperometric measures of NO representing the constitutive isoforms of neuronal nitric oxide synthase (nNOS) and endothelial (e)NOS were also taken at each time point to ascertain whether NO levels changed as a result of CBH, and if so, whether such NO changes preceded or followed any memory or amyloid-beta pathology. We found that two weeks after CBH, NO hippocampal levels were upregulated nearly four-fold when compared to nonoccluded rats but no alteration in spatial memory of A beta products were observed at this time point. By contrast, NO concentration had declined to control levels by eight weeks but spatial memory was found significantly impaired and A beta 1-40 (but not A beta 1-42) had increased in the CBH group when compared to control rats. Since changes in shear stress are known to upregulate eNOS but generally not nNOS, these results suggest that shear stress induced by CBH hyperactivated vascular NO derived from eNOS in the first two weeks as a reaction by the capillary endothelium to maintain homeostasis of local cerebral blood flow. The return of vascular NO to basal levels after eight weeks of CBH may have triggered metabolic changes within hippocampal cells resulting in hippocampal dysfunction as reflected by spatial memory impairment and by accumulation of A beta 1-40 peptide. In conclusion, our study shows that CBH initiates spatial memory loss in aging rats thus mimicking human MCI and also increases A beta 1-40 in the hippocampus. The memory and amyloid changes are preceded by NO upregulation in the hippocampus. These preliminary findings may be important in understanding, at least in part, the molecular mechanisms that precede memory impairment during chronic brain ischemia and as such, the pre-clinical stage leading to Alzheimer's disease.

Differentiation and distribution of acetylcholinesterase molecular forms in the mouse cochlea.

The activities of the globular and asymmetric forms of acetylcholinesterase (AChE) were measured in the whole cochlea and cochlear turns of the developing postnatal mouse. The globular AChE forms (G4, G2 and G1) were present in each cochlear turn at birth. An asymmetric AChE form (A12) was detected in the midturn on the 4th postnatal day, and in the base and apex on the 7th postnatal day. The activities of all AChE molecular forms increased rapidly during the second postnatal week and reached a plateau by the 19th postnatal day. In the 26-day old mouse, G4 constitutes the largest proportion of total cochlear AChE (57%), G2/G1 being 37% and A12 being 6%. The distribution of the AChE forms among the different turns is as follows: the combined value of the activities of G2 and G1 AChE was the same in each turn; G4 was the major form in the base and midturn; and A12, the least abundant AChE form of all, was localized mainly in the base. Our results indicate that in the cochlea (1) the content of molecular forms is similar to that of other neuronal systems, (2) the expression of AChE molecular forms is developmentally regulated, and (3) the AChE isoenzymes develop and are distributed differentially along the cochlear length; resulting near maturity in the greater proportional expression of G4 and A12 in the base and midturn and G2/G1 in the apex.

Acetylcholinesterase-positive innervation in cochleas from two strains of shaker-1 mice.

Shaker-1 is a recessive gene mutation on chromosome 7 in mice, causing both deafness and neurosensory degeneration in the inner ear. A failure of efferent innervation to the outer hair cells is being implicated in the cause of deafness (Green, 1981). To investigate the efferent innervation, we examined the cochleas of two strains of shaker-1 mutants: Sh1/Le (25 and 45 days old) and FS/Ei (28 and 68 days old), using enzymatic staining of acetylcholinesterase (AChE) for the light and electron microscopes, and also by measuring the activities of AChE and of AChE molecular forms. The enzyme levels in the SH1/Le and FS/Ei homozygotes (sh-1/sh-1) were within the range of those in SH1/Le heterozygotes (+/sh-1) and in normal mouse strains (C3H/HeJ, 129/SvJ, ICR). The picture of AChE-positive innervation in both strains differed. In the SH1/Le mutants at 25 days, the innervation appeared normal, but by 45 days it showed a marked atrophy. In the FS/Ei mutants, the degeneration was already evident by the 28th day. In the younger animals of both mutants, large differentiated vesiculated nerve endings were ultrastructurally detected in synaptic contact with outer hair cells. The preservation of AChE activity and of the expression of AChE molecular forms up to 68 days indicate that the shaker-1 cochlea may initially possess a normal input of AChE-positive efferent innervation. The late onset and the slow course of the degeneration of AChE-positive innervation seen in the SH1/Le mutants suggest that the loss of efferent endings may be, contrary to previous suggestions, the consequence rather than the cause of the shaker-1 pathology.