Isolation and chemical characterization of Alzheimer's disease paired helical filament cytoskeletons: differentiation from amyloid plaque core protein.

The paired helical filaments (PHFs) of Alzheimer's disease were purified by a strategy in which the neurons and amyloid plaque cores of protein (APCP) were initially isolated. This was achieved by several steps of isocratic sucrose centrifugations of increasing molarity and a discontinuous isotonic Percoll density gradient. After collagenase elimination of contaminating blood vessels, lysis of neurons was produced by SDS treatment. The released PHF cytoskeletons were separated from contaminating APCP and lipofuscin by sucrose density gradient. A final step consisted in the chemical purification of highly enriched PHFs and APCP components via a formic acid to guanidine hydrochloride transition. PHFs and APCPs were fractionated by size exclusion HPLC and further characterized and quantitated by automatic amino acid analysis. We also present some of the morphological and immunochemical characteristics of PHF polypeptides and APCP. Our studies indicate that apart from differences in localization and morphology, PHF and APCP significantly differ in (a) chemical structure (peptide and amino acid composition); (b) epitope specificity (antiubiquitin, antitau, antineurofilament); (c) physicochemical properties (structural conformation in guanidine hydrochloride); and (d) thioflavine T fluorescence emission. These parameters strongly suggest important differences in the composition and, probably, in the etiopathology of PHF and APCP of Alzheimer's disease.

Disorders of learning and memory processes in a monkey model of Alzheimer's disease: the role of the associative area of the cerebral cortex.

The processes of learning and storage of the results of learning were studied in a model of Alzheimer's disease in two groups of rhesus macaques (three individuals in each group). Studies were performed after injection of neurotoxins (group I) and physiological saline (group II, controls). Two months after injections (stage C1), learning parameters were studied in monkeys of both groups using a new stimulus discrimination test (filled geometrical figures versus outline figures). There were significant differences between the animals of the two groups. Learning was hindered in monkeys of group I, with significant increases in the learning time (the time to achieve a stable probability of correct responding of 0.85) and in the probability of refusals. Monkeys of group II showed no learning impairment. Animals were trained to discriminate new stimuli (images of two monkeys) six months after injections (stage C3). Learning was impaired in animals of group I, such that learning measures had the same levels as previously; monkeys of group II showed no learning impairment. Analysis of the characteristics of working memory, which is involved in storing the results of new learning, was performed at stage C1; monkeys of group I showed significant degradation of these measures, with a significant decrease in the probability of correct solutions at stage C1 (to a level of 0.5), with some increase at stages C2 (at four months) and C3, along with a significant increase in the probability of refusals, values being similar at all time points. For monkeys of group II, these characteristics showed no degradation. Motor response times at stages C1, C2, and C3 were not different for the two groups of monkeys. The structural-functional organization of interactions between sensory and cognitive processes during learning and the storage of information in working memory are discussed, as is the role of the associative areas of the cortex in these interactions.

Evidence for seeding of beta -amyloid by intracerebral infusion of Alzheimer brain extracts in beta -amyloid precursor protein-transgenic mice.

Many neurodegenerative diseases are associated with the abnormal sequestration of disease-specific proteins in the brain, but the events that initiate this process remain unclear. To determine whether the deposition of the beta-amyloid peptide (Abeta), a key pathological feature of Alzheimer's disease (AD), can be induced in vivo, we infused dilute supernatants of autopsy-derived neocortical homogenates from Alzheimer's patients unilaterally into the hippocampus and neocortex of 3-month-old beta-amyloid precursor protein (betaAPP)-transgenic mice. Up to 4 weeks after the infusion there was no Abeta-deposition in the brain; however, after 5 months, the AD-tissue-injected hemisphere of the transgenic mice had developed profuse Abeta-immunoreactive senile plaques and vascular deposits, some of which were birefringent with Congo Red. There was limited deposition of diffuse Abeta also in the brains of betaAPP-transgenic mice infused with tissue from an age-matched, non-AD brain with mild beta-amyloidosis, but none in mice receiving extract from a young control case. Abeta deposits also were not found in either vehicle-injected or uninjected transgenic mice or in any nontransgenic mice. The results show that cerebral beta-amyloid can be seeded in vivo by a single inoculation of dilute AD brain extract, demonstrating a key pathogenic commonality between beta-amyloidosis and other neurodegenerative diseases involving abnormal protein polymerization. The paradigm can be used to clarify the conditions that initiate in vivo beta-amyloidogenesis in the brain and may yield a more authentic animal model of Alzheimer's disease and other neurodegenerative disorders.

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.

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.

Protein aging Extracellular amyloid formation and intracellular repair.

Soluble proteins can undergo spontaneous structural and conformational alterations that lead to their stable aggregation into amyloid fibrils. Amyloidogenic proteins have been implicated in several types of age-related pathologic changes. For example, transthyretin amyloid accumulation in the heart can lead to cardiac failure, while ß-amyloid deposition within the microvasculature and gray matter of the brain is linked to cerebral hemorrhage and neuronal death. Over the course of evolution, protein structures have developed that largely resist such aggregation. Spontaneous chemical modifications correlated with the normal aging process, however, including the deamidation, isomerization, and racemization of asparaginyl and aspartyl residues, as well as the oxidation and glycation of various amino acid residues, may contribute to amyloid formation by altering protein structure. In fact, a recent chemical analysis of neuritic plaque and vascular ß-amyloid deposits from the brains of Alzheimer's disease victims has revealed that the majority of the aspartyl residues in ß-amyloid are in the isomerized and/or racemized configuration. Although enzymes exist that can reverse at least part of this damage for intracellular proteins, the accumulation of extracellular proteins containing altered residues might contribute to the deterioration of heart, brain, and other tissues that occurs with aging and disease.

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.

Increased accumulation of cAMP in cerebral microvessels in Alzheimer's disease.

The objective of this study was to compare the adenylyl cyclase-cAMP second messenger system in cerebral microvessels isolated from Alzheimer's patients to that in microvessels from nondemented elderly controls. To evaluate the role of aging separate from the effects of dementia, microvessels from young and aged rodents were also examined. The results of this study indicated that microvessels isolated from autopsy material can be used to evaluate adenylyl cyclase activity. The data showed that cAMP levels, as an index of adenylyl cyclase activity, are significantly (p < 0.02) elevated in microvessels from Alzheimer's disease compared to nondemented elderly controls. Stimulation of adenylyl cyclase by forskolin was comparable in both groups of microvessels. A comparison of unstimulated microvessels from young and aged rodents yielded no significant difference in cAMP levels. These results indicate an increased level of cAMP in the microvessels of Alzheimer's patients with no age-related change demonstrable in rat microvessels.

RAGE-Abeta interactions in the pathophysiology of Alzheimer's disease.

RAGE is a cell surface molecule primarily identified for its capacity to bind advanced glycation end-products and amphoterin. Immunocytochemical studies demonstrated that in Alzheimer's Disease (AD) the expression of RAGE is elevated in neurons close to neuritic plaque beta-amyloid (Abeta) deposits and in the cells of Abeta containing vessels. Cross-linking of surface bound Abeta 1-40 to endothelial cells, yielded a band of 50 kDa identified as RAGE. Using the soluble extracellular domain of recombinant human RAGE, we found that Abeta binds to RAGE with a Kd = 57 +/- 14 nM, a value close to those found for mouse brain endothelial cells and rat cortical neurons. The interaction of Abeta with RAGE in neuronal, endothelial, and RAGE-transfected COS-1 cells induced oxidative stress, as assessed by the TBARS and MTT assays. ELISA demonstrated a 2.5 times increase of RAGE in AD over control brains. Activated microglia also showed elevated expression of RAGE. In the BV-2 microglial cell line, RAGE bound Abeta in dose dependent manner with a Kd of 25 +/- 9 nM. Soluble Abeta induced the migration of microglia along a concentration gradient, while immobilized Abeta arrested this migration. Abeta-RAGE interaction also activated NF-kappaB, resulting in neuronal up-regulation of macrophage-colony stimulating factor (M-CSF) which also induced microglial migration. Taken together, our data suggest that RAGE-Abeta interactions play an important role in the pathophysiology of Alzheimer's Disease.

Cerebral amyloid angiopathy: accumulation of A beta in interstitial fluid drainage pathways in Alzheimer's disease.

Cerebral amyloid angiopathy (CAA) is characterized by the accumulation of beta-amyloid (A beta) peptides in the walls of arteries both in the cortex and meninges. Here, we test the hypothesis that CAA results from the progressive accumulation of A beta in the perivascular interstitial fluid drainage pathways of the brain. Experimental studies have shown that interstitial fluid (ISF) from the rat brain flows along periarterial spaces to join the cerebrospinal fluid (CSF) to drain to cervical lymph nodes. Such lymphatic drainage plays a key role in B-cell and T-cell mediated immunity of the brain. Anatomical studies have defined periarterial ISF drainage pathways in the human brain that are homologous with the lymphatic pathways in the rat brain but are largely separate from the CSF. Periarterial channels in the brain in man are in continuity with those of leptomeningeal arteries and can be traced from the brain to the extracranial portions of the internal carotid arteries related to deep cervical lymph nodes. The pattern of deposition of A beta in senile plaques and in CAA suggests that A beta accumulates in pericapillary and periarterial ISF drainage pathways. A beta could accumulate in CAA due to either (i) increased production of A beta, (ii) reduced solubility of A beta peptides, or (iii) impedance of drainage of A beta along periarterial ISF drainage pathways within the brain and leptomeninges due to aging factors in cerebral arteries. Elucidation of factors that reduce elimination of A beta via perivascular drainage pathways may lead to their rectification and to new strategies for treatment of Alzheimer's disease.

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.

Reduction of cortical amyloid beta levels in guinea pig brain after systemic administration of physostigmine.

Overproduction of the peptide amyloid beta (Abeta) is thought to be a critical pathogenetic event in Alzheimer's disease (AD). Decreasing A production may therefore slow or halt the progression of AD. In vitro work has indicated that cholinergic muscarinic receptor agonists may reduce cellular production of Abeta. Here we show that systemic administration of physostigmine, an acetylcholinesterase inhibitor, lowers Abeta levels in vivo. Guinea pigs treated for 10 days with s.c. physostigmine had levels of cortical AbetaN-40 and N-42 which were 57% and 72%, respectively, of those in control animals. Levels of cortical beta-amyloid precursor protein were not significantly affected by drug treatment. These results suggest that cholinergic therapy may affect the course of AD by limiting Abeta accumulation.

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.

New biochemical insights to unravel the pathogenesis of Alzheimer's lesions.

Purification of amyloid plaque core proteins (APCP) from Alzheimer's disease brains to complete homogeneity and in high yield permitted its chemical fractionation and characterization of its components. APCP is mainly made of beta-amyloid (beta A) and an assortment of glycoproteins (accounting for 20%) rich in carbohydrates compatible with N- and O-linked saccharides. When added to tissue culture of sympathetic and sensory neurons APCP and beta A inhibited neuritic sprouting, a reversible phenomenon at low doses. Higher concentrations of both substances kill the neurons in culture. APCP is significantly more toxic than beta A, suggesting the minor components may play an important role in increasing the toxicity of beta A. If the observed toxic effects of APCP in situ are occurring in vivo during the course of AD, then the accumulation of these extracellular proteins could be largely responsible for some of the neuronal death observed in this neuropathology.

[Disorders in learning and memory processes in the monkey model of Alzheimer's disease: the role of the cerebral cortex associative areas].

Processes of novelty learning and keeping the results in Alzheimer's disease in two groups of rhesus-monkeys (three monkeys in each group), were studied: following neurotoxins administrati- on (I group) and saline administration (II group). In two months after the injections (the C1 stage), considerable differences between the groups were revealed in the task of differentiation among contour shapes. For the I group monkeys the learning was difficult: the correct decision making did not reach 85 %, and the probability of refusing to make a decision increased. For the II group monkeys the learning characteristics were not disturbed. In six months after the injections (the C3 stage) the differences between the groups in the task of differentiation among new stimuli (heads of two monkeys) remained at the same level. When studying characteristics of the operative memory associated with keeping the learning results achieved at the C1 stage, a considerable worsening of these characteristics was revealed: diminishing of the correct decision making probability at the C1 stage (actually to the level of 0.5), increase in the probability of refusing to make a decision. The structural-functional organization of interaction between sensory and cognitive processes in learning and keeping the information in the operative memory, is discussed in association with the control of motivation and attention system and the role of the cortex associative areas.