Systemic catabolism of Alzheimer's Abeta40 and Abeta42

To better understand the physiologic excretion and/or catabolism of circulating peripheral amyloid beta (Abeta), we labeled human Abeta40 (monomeric, with predominant unordered structure) and Abeta42 (mixture of monomers and oligomers in approximately 50:50 ratio, rich in beta-sheet conformation) with either Na(125)I or (125)I-tyramine cellobiose, also known as the cell-trapping ligand procedure, testing their blood clearance and organ uptake in B6SJLF1/J mice. Irrespective of the labeling protocol, the peptide conformation, and the degree of oligomerization, both Abeta40 and Abeta42 showed a short half-life of 2.5-3.0 min. The liver was the major organ responsible for plasma clearance, accounting for >60% of the peptide uptake, followed by the kidney. In vivo, hepatocytes captured >90% of the radiolabeled peptides which, after endocytosis, were preferentially catabolized and excreted into the bile. Biliary excretion of intact as well as partially degraded Abeta species became obviously relevant at doses above 10 microg. The use of biotin-labeled Abeta allowed the visualization of the interaction with HepG2 cells in culture, whereas competitive inhibition experiments with unlabeled Abeta demonstrated the specificity of the binding. The capability of the liver to uptake, catabolize, and excrete large doses of Abeta, several orders of magnitude above its physiologic concentration, may explain not only the femtomolar plasma levels of Abeta but the little fluctuation observed with age and disease stages.     

Histidines 13 and 14 in the Abeta sequence are targets for inhibition of Alzheimer's disease Abeta ion channel and cytotoxicity

The fact that Alzheimer's beta amyloid (Abeta) peptides forms cation channels in lipid bilayers was discovered during the course of our experiments in the laboratory of "Guayo" Rojas at NIH in Bethesda, Maryland (USA). Recently, we found that the Abeta ion channel could be blocked selectively with small peptides that copy the amino acid sequence of the predicted mouth region of the Abeta channel pore. We now have searched for the essential amino acid residues required for this blocking effect by mutations. We found that the ability of peptides to block Abeta channel activity could be lost by replacement of histidines 13 and 14 by alanine or lysine. The amino acid substitution also resulted in the loss of the capacity of the peptides to protect cells from Abeta cytotoxicity. These data thus contribute to the definition of the region of the Abeta sequence that participates in the formation of the channel pore. Additionally, these data support the hypothesis that the ion channel activity of Ab contributes significantly to the cytotoxic properties of Abeta. These data also emphasize the potential value in using inhibition of Abeta ion channel activity as an end point for Alzheimer's disease drug discovery.     

CLAC binds to aggregated Abeta and Abeta fragments, and attenuates fibril elongation

Deposition of amyloid beta-peptide (Abeta) into amyloid plaques is one of the invariant neuropathological features of Alzheimer's disease. Proteins that codeposit with Abeta are potentially important for the pathogenesis, and a recently discovered plaque-associated protein is the collagenous Alzheimer amyloid plaque component (CLAC). In this study, we investigated the molecular interactions between Abeta aggregates and CLAC using surface plasmon resonance spectroscopy and a solid-phase binding immunoassay. We found that CLAC binds to Abeta with high affinity, that the central region of Abeta is necessary and sufficient for CLAC interaction, and that the aggregation state of Abeta as well as the presence of negatively charged residues is important. We also show that this binding results in a reduced rate of fibril elongation. Taken together, we suggest that CLAC becomes involved at an intermediate stage in the pathogenesis by binding to Abeta fibrils, including fibrils formed from peptides with truncated N- or C-termini, and thereby slows their growth.     

Linking calcium to Abeta and Alzheimer's disease

Recent developments point to a critical role for calcium dysregulation in the pathogenesis of Alzheimer's disease. A novel calcium-conducting channel called CALHM1 is genetically linked to the disorder and modulates Abeta production. Calcium homeostasis has also been shown to be perturbed in dendritic spines adjacent to amyloid plaques. Finally, new studies have elucidated the role by which presenilins modulate calcium signaling, including effects on SERCA2b and gating of the IP(3) receptor, and lead to Abeta production.     

Dyrk1 inhibition improves Alzheimer's disease‐like pathology

There is an urgent need for the development of new therapeutic strategies for Alzheimer's disease (AD). The dual‐specificity tyrosine phosphorylation‐regulated kinase‐1A (Dyrk1a) is a protein kinase that phosphorylates the amyloid precursor protein (APP) and tau and thus represents a link between two key proteins involved in AD pathogenesis. Furthermore, Dyrk1a is upregulated in postmortem human brains, and high levels of Dyrk1a are associated with mental retardation. Here, we sought to determine the effects of Dyrk1 inhibition on AD‐like pathology developed by 3xTg‐AD mice, a widely used animal model of AD. We dosed 10‐month‐old 3xTg‐AD and nontransgenic (NonTg) mice with a Dyrk1 inhibitor (Dyrk1‐inh) or vehicle for eight weeks. During the last three weeks of treatment, we tested the mice in a battery of behavioral tests. The brains were then analyzed for the pathological markers of AD. We found that chronic Dyrk1 inhibition reversed cognitive deficits in 3xTg‐AD mice. These effects were associated with a reduction in amyloid‐β (Aβ) and tau pathology. Mechanistically, Dyrk1 inhibition reduced APP and insoluble tau phosphorylation. The reduction in APP phosphorylation increased its turnover and decreased Aβ levels. These results suggest that targeting Dyrk1 could represent a new viable therapeutic approach for AD.     

Abeta deposition and related pathology in an APP x PS1 transgenic mouse model of Alzheimer's disease

A transgenic mouse bearing mutant transgenes linked to familial forms of Alzheimer's disease (AD) for the amyloid precursor protein and presenilin-1 (TASTPM) showed Abeta plaque deposition and age-related histological changes in associated brain pathology. The Abeta present was of multiple forms, including species with a C-terminus at position 40 or 42, as well as an N-terminus at position 1 or truncated in a pyro-3-glutamate form. Endogenous rodent Abeta was also present in the deposits. Laser capture microdissection extracts showed that multimeric forms of Abeta were present in both plaque and tissue surrounding plaques. Associated with the Abeta deposits was evidence of an inflammatory response characterised by the presence of astrocytes. Also present in close association with the deposits was phosphorylated tau and cathepsin D immunolabelling. The incidence of astrocytes and of phosphorylated tau and cathepsin D load showed that both of these potential disease markers increased in parallel to the age of the mice and with Abeta deposition. Immunohistochemical labelling of neurons in the cortex and hippocampus of TASTPM mice suggested that the areas of Abeta deposition were associated with the loss of neurons. TASTPM mice, therefore, exhibit a number of the pathological characteristics of disease progression in AD and may provide a means for assessment of novel therapeutic agents directed towards modifying or halting disease progression.     

Modulation of Alzheimer's pathology by cerebro-ventricular grafting of hybridoma cells expressing antibodies against Abeta in vivo

Accumulation in brain of the beta-amyloid peptide (Abeta) is considered as crucial pathogenic event causing Alzheimer's disease (AD). Anti-Abeta immune therapy is a powerful means for Abeta clearance from the brain. We recently showed that intravenous injections of anti-Abeta antibodies led to reduction, elevation or no change in brain Abeta42 concentrations of an AD mouse model. We report here, in a second passive immunization protocol, a different bioactivity of same antibodies to alter brain Abeta42 concentrations. Comparing the bioactivity of anti-Abeta antibodies in these two passive immunization paradigms underscores the potential of immune therapy for AD treatment and suggests that both the epitope recognized by the antibody and the mode of antibody administration are crucial for its biological activity.     

Clathrin-mediated endocytic proteins are upregulated in the cortex of the Tg2576 mouse model of Alzheimer's disease-like amyloid pathology

Amyloid-β (Aβ) is cleaved from amyloid precursor protein (APP) predominantly after APP has trafficked through the secretory pathway and then become re-internalised by endocytosis. Clathrin-mediated and, more recently, clathrin-independent endocytosis have both been implicated in this process. Furthermore, endocytic abnormalities have been identified in cases of Alzheimer’s disease (AD), however, the relevance of these changes to the aetiology of the disease remains unclear. We therefore examined the expression of proteins related to these endocytic processes in the cortex of Tg2576 mice that overexpress the Swedish mutation in APP, and consequently overexpress Aβ, to determine if there were any changes in their associated pathways. We identified significant increases in the levels of clathrin, dynamin and PICALM, all proteins intimately involved with the clathrin-mediated endocytic pathway, in the transgenic animals. However, levels of proteins associated with flotillin or caveolin-mediated endocytic pathways remained unchanged. These results emphasise the importance of clathrin-mediated endocytosis in the aetiology of AD and reinforce the results of the recent GWAS studies that identified genes for clathrin-mediated endocytosis as susceptibility genes for AD. Such studies in transgenic mice will allow us to learn more about the role of clathrin-mediated endocytosis in AD.     

Amiloride interacts with guanine nucleotide regulatory proteins and attenuates the hormonal inhibition of adenylate cyclase

The effect of amiloride on the hormonal regulation of adenylate cyclase was studied in the rat anterior pituitary. The diuretic did not alter basal adenylate cyclase but augmented the enzyme activity in an irreversible manner in the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S) stimulated adenylate cyclase at lower concentrations and inhibited at higher concentrations. Amiloride treatment enhanced the stimulatory and abolished the inhibitory phase of GTP gamma S action. In addition, amiloride also attenuated the inhibitory effects of atrial natriuretic factor (ANF 99-126) and angiotensin II on cAMP levels and adenylate cyclase activity. On the other hand, amiloride showed an additive effect on the stimulation exerted by corticotropin-releasing factor and vasoactive intestinal peptide on adenylate cyclase in anterior pituitary and on isoproterenol-stimulated cAMP levels in cultured vascular smooth muscle cells. Pertussis toxin, in the presence of [alpha-32 P]NAD, catalyzed the ADP-ribosylation of two protein bands of Mr 41,000 and 39,000, referred to as Gi and Go, respectively, in the anterior pituitary, and 40,000-Da protein in the aorta, referred to as Gi. Amiloride treatment inhibited the labeling of all these bands in a concentration- and time-dependent manner. Similarly, the pertussis toxin-catalyzed ADP-ribosylation of purified Gi from bovine brain was also inhibited by amiloride treatment. However, amiloride had no significant effect on the cholera toxin-catalyzed ADP-ribosylation of Gs. These data suggest that amiloride interacts with the guanine nucleotide regulatory proteins Gi and Go. Modification of Gi results in the attenuation of hormone-induced adenylate cyclase and cAMP inhibition. However, the interaction between amiloride and Go and the consequent Ca2+ mobilization and phosphatidylinositol turnover have to be investigated.     

Alzheimer's disease: Abeta, tau and synaptic dysfunction

Alzheimer's disease is a progressive neurodegenerative disorder that is characterized by two hallmark lesions: extracellular amyloid plaques and neurofibrillary tangles. The role that these lesions have in the pathogenesis of AD has proven difficult to unravel, in part because of unanticipated challenges of reproducing both pathologic hallmarks in transgenic mice. Recent advances in recapitulating both plaques and tangles in the brains of transgenic mice are leading to novel insights into their role in the degenerative process, including their impact on synaptic activity and plasticity. Transgenic mice that harbor both neuropathological lesions are also facilitating the elucidation of the relationship of these proteinaceous aggregates to one another and providing a crucial in vivo system for developing and evaluating therapies.     

Biogenesis and metabolism of Alzheimer's disease Abeta amyloid peptides

Biochemical and genetic evidence indicates the balance of biogenesis/clearance of Abeta amyloid peptides is altered in Alzheimer's disease. Abeta is derived, by two sequential cleavages, from the receptor-like amyloid precursor protein (APP). The proteases involved are beta-secretase, identified as the novel aspartyl protease BACE, and gamma-secretase, a multimeric complex containing the presenilins (PS). Gamma-secretase can release either Abeta40 or the more aggregating and cytotoxic Abeta42. Secreted Abeta peptides become either degraded by the metalloproteases insulin-degrading enzyme (IDE) and neprilysin or metabolized through receptor uptake mediated by apolipoprotein E. Therapeutic approaches based on secretase inhibition or amyloid clearance are currently under development.     

Clearing tau pathology with Abeta immunotherapy--reversible and irreversible stages revealed

The report by Oddo and colleagues in this issue of Neuron demonstrates for the first time that clearance of amyloid also results in the removal of early-stage tau pathology in mice that develop both amyloid plaques and neurofibrillary tangles (NFT), the two hallmark lesions of Alzheimer's disease (AD). This result supports a primary role for Abeta in AD etiology.     

Glutaminyl cyclase knock-out mice exhibit slight hypothyroidism but no hypogonadism: implications for enzyme function and drug development

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) residues at the N terminus of peptides and proteins. Hypothalamic pGlu hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone are essential for regulation of metabolism and fertility in the hypothalamic pituitary thyroid and gonadal axes, respectively. Here, we analyzed the consequences of constitutive genetic QC ablation on endocrine functions and on the behavior of adult mice. Adult homozygous QC knock-out mice are fertile and behave indistinguishably from wild type mice in tests of motor function, cognition, general activity, and ingestion behavior. The QC knock-out results in a dramatic drop of enzyme activity in the brain, especially in hypothalamus and in plasma. Other peripheral organs like liver and spleen still contain QC activity, which is most likely caused by its homolog isoQC. The serum gonadotropin-releasing hormone, TSH, and testosterone concentrations were not changed by QC depletion. The serum thyroxine was decreased by 24% in homozygous QC knock-out animals, suggesting a mild hypothyroidism. QC knock-out mice were indistinguishable from wild type with regard to blood glucose and glucose tolerance, thus differing from reports of thyrotropin-releasing hormone knock-out mice significantly. The results suggest a significant formation of the hypothalamic pGlu hormones by alternative mechanisms, like spontaneous cyclization or conversion by isoQC. The different effects of QC depletion on the hypothalamic pituitary thyroid and gonadal axes might indicate slightly different modes of substrate conversion of both enzymes. The absence of significant abnormalities in QC knock-out mice suggests the presence of a therapeutic window for suppression of QC activity in current drug development.     

Direct observation of Abeta amyloid fibril growth and inhibition

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.     

Focal adhesions regulate Abeta signaling and cell death in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder that results from a loss of synaptic transmission and ultimately cell death. The presenting pathology of AD includes neuritic plaques composed of beta-amyloid peptide (Abeta) and neurofibrillary tangles composed of hyperphosphorylated tau, with neuronal loss in specific brain regions. However, the mechanisms that induce neuronal cell loss remain elusive. Focal adhesion (FA) proteins assemble into intracellular complexes involved in integrin-mediated communication between the extracellular matrix and the actin cytoskeleton, regulating many cell physiological processes including the cell cycle. Interestingly, recent studies report that integrins bind to Abeta fibrils, mediating Abeta signal transmission from extracellular sites of Abeta deposits into the cell and ultimately to the nucleus. In this review, we will discuss the Abeta induced integrin/FA signaling pathways that mediate cell cycle activation and cell death.     

beta-site specific intrabodies to decrease and prevent generation of Alzheimer's Abeta peptide

Endoproteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases generates the toxic amyloid beta-peptide (Abeta), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Abeta based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the beta-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the beta-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by alpha-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Abeta. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.     

Pertussis toxin attenuates atrial natriuretic factor-mediated inhibition of adenylate cyclase. Involvement of inhibitory guanine nucleotide regulatory protein

The effect of pertussis toxin treatment was studied on the inhibitory effect of atrial natriuretic factor (ANF) on adenylate cyclase activity in rat aorta. The incubation of rat aorta washed particles with pertussis toxin and [alpha-32P]NAD resulted in the ADP-ribosylation of a single 40-kDa protein. In addition, pertussis toxin treatment enhanced guanosine 5'-O-(thiotriphosphate) and isoproterenol-stimulated adenylate cyclase activities and attenuated the ANF-mediated inhibition of basal, isoproterenol-, and forskolin-stimulated adenylate cyclase activities. These data suggest that ANF receptors are coupled to adenylate cyclase through inhibitory guanine nucleotide regulatory protein.     

Intra- and extracellular Abeta and PHF in clinically evaluated cases of Alzheimer's disease

Temporal cortical sections from postmortem brains of individuals without any dementing condition and with different degrees of severity of Alzheimer's disease (AD) evaluated by the Clinical Dementia Rating scale (CDR 0-CDR 3) were analyzed using immunohistochemical procedures. To demonstrate the amyloid-beta-peptide (Abeta) deposition and the neurofibrillary pathology, two monoclonal antibodies were used, a human CERAD Abeta (10D5) antibody raised against the N-terminal region of the Abeta-peptide, and an antibody raised against paired helical filaments (PHF-1). The neuron cell bodies and the glial cells were also recognized by two polyclonal antibodies raised, respectively, against the protein gene peptide (PGP 9.5) and glial fibrillary acidic protein (GFAP). Directly related to severity of AD, progressive deposits of Abeta-peptide were found within cortical pyramidal-like neurons and forming senile plaques. Ultrastructurally, Abeta-peptide deposits were related to neuronal intracytoplasmic organelles, such as the ER, the mitochondria, the Nissl bodies and lipofuscin. We have also found that the intracellular deposition of the Abeta peptide is a neuropathological finding prior to the appearance of PHF-immunoreactive structures. We suggest that the intracellular Abeta deposition in cortical pyramidal neurons is a first neurodegenerative event in AD development and that it is involved in cell dysfunction, neuronal death, and plaque formation.