Endocrine Involvement in Systemic Amyloidosis

ObjectiveTo present an overview of the published data on endocrine involvement and endocrine dysfunction in patients with systemic amyloidosis.MethodsWe conducted a review of the medical literature using MEDLINE data sources, including clinical trials, in vitro studies, and case reports on pituitary, thyroid, parathyroid, pancreatic, adrenal, and gonadal involvement in systemic amyloidosis.ResultsReports of endocrine involvement in systemic amyloidosis seem to consist primarily of small-samplesize clinical trials or case reports, probably because of the rarity of the disease itself. Systemic amyloidosis mainly involves and causes functional impairment in the thyroid and testes in the endocrine system. Evaluation of adrenal function necessitates special consideration because amyloid infiltration of the adrenal glands resulting in failure may be a life-threatening condition. Amyloid deposition commonly seen in the pituitary gland and the pancreas of patients with Alzheimer disease and type 2 diabetes mellitus, respectively, is generally classified as local amyloidosis and should not be confused with systemic involvement. Additionally, detection of amyloid deposition in the thyroid and testes may have a diagnostic role in patients with suspected systemic or renal amyloidosis.ConclusionPublished data suggest that systemic amyloidosis frequently involves the endocrine system, and endocrine dysfunction seems to be not as rare as previously thought. A rapidly growing goiter or symptoms and signs of adrenal or gonadal dysfunction should raise suspicion of amyloid infiltration. Involvement of pituitary, parathyroid, and pancreatic sites in systemic amyloidosis still remains to be clarified. Further studies with larger sample sizes are needed for complete characterization of the effect of systemic amyloidosis on the endocrine system. (Endocr Pract. 2010;16:1056-1063)     

Template-directed self-assembly and growth of insulin amyloid fibrils

The formation of amyloid aggregates in tissue is a pathological feature of many neurodegenerative diseases and type II diabetes. Amyloid deposition, the process of amyloid growth by the association of individual soluble amyloid molecules with a pre-existing amyloid template (i.e., plaque), is known to be critical for amyloid formation in vivo. The requirement for a natural amyloid template, however, has made amyloid deposition study difficult and cumbersome. In the present work, we developed a novel, synthetic amyloid template by attaching amyloid seeds covalently onto an N-hydroxysuccinimide-activated surface, where insulin was chosen as a model amyloidogenic protein. According to ex situ atomic force microscopy observations, insulin monomers in solution were deposited onto the synthetic amyloid template to form fibrils, like hair growth. The fibril formation on the template occurred without lag time, and its rate was highly accelerated than in the solution. The fibrils were long, over 2 mum, and much thinner than those in the solution, which was caused by limited nucleation sites on the template surface and lack of lateral twisting between fibrils. According to our investigations using thioflavin T-induced fluorescence, birefringent Congo red binding, and circular dichroism, fibrils grown on the template were identified to be amyloids that formed through a conformational rearrangement of insulin monomers upon interaction with the template. The amyloid deposition rate followed saturation kinetics with respect to insulin concentration in the solution. The characteristics of amyloid deposition on the synthetic template were in agreement with previous studies performed with human amyloid plaques. It is demonstrated that the synthetic amyloid template can be used for the screening of inhibitors on amyloid deposition in vitro.     

Differential expression of the amyloid SAA 3 gene in liver and peritoneal macrophages of mice undergoing dissimilar inflammatory episodes

The three active serum amyloid A (SAA) genes of mice, SAA 1, SAA 2, and SAA 3, are coordinately expressed in liver during acute and chronic inflammatory stimulation and experimental amyloidosis. The genes, primarily SAA 3, are also expressed extrahepatically. The apoprotein SAA 2 is the precursor of the amyloid A (AA) fibril protein that is deposited as insoluble fibrils extracellularly in spleen and other organs when amyloidosis occurs secondarily to inflammation. The exact cause of AA fibril formation is unknown. Amyloid enhancing factor is a high m.w. glycoprotein extracted from amyloidotic organs. Administration of amyloid enhancing factor alters experimental inflammation to bring about accelerated deposition of amyloid A fibrils first in spleen and later in other organs. In this study, hepatic and extrahepatic expression of the SAA genes were compared during accelerated amyloidosis relative to inflammation uncomplicated by amyloidosis. Differences in kinetics and pattern of SAA gene expression by resident peritoneal macrophages and liver were detected during four dissimilar inflammatory episodes. Macrophages expressed the SAA 3 gene solely, and to a greater extent in chronic than in acute inflammation. In accelerated amyloid induction, macrophage SAA 3 expression increased as SAA 1 and SAA 2 expression in liver decreased. However, alpha-1-acid glycoprotein expression remained elevated throughout the course of amyloid induction. The greatly increased expression of the SAA 3 gene by macrophages and decreased expression of the SAA 1 and SAA 2 genes in liver during amyloidosis, suggests that altered SAA gene expression may play a pathogenetic role in experimental amyloid deposition.     

Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met 30) gene

To analyze the pathologic processes of amyloid deposition in type I familial amyloidotic polyneuropathy (FAP), mice were made transgenic by introducing the human mutant transthyretin (TTR) gene(MT-hMet 30). An inbred strain of mouse, C57 BL/6, was chosen. Transgenic mice were killed using ether anesthesia at 3-mo intervals up to 24 mo after birth. In these transgenic mice, amyloid deposition started in the gastrointestinal tract, cardiovascular system, and kidneys and extended to various other organs and tissues with advancing age. The pattern of amyloid deposition was similar to that observed in human autopsy cases of FAP, except for its absence in the choroid plexus and in the peripheral and autonomic nervous systems. We extracted the amyloid fibrils from kidneys of these mice with a human mutant TTR gene and analyzed them immunochemically and electronmicroscopically. Deposited amyloid was shown to be composed of human mutant TTR and mouse serum amyloid P component. Amyloid fibril from transgenic mice was morphologically and immunohistochemically similar to that of human FAP. The most striking pathologic feature of the transgenic mice was the absence of amyloid deposition in the peripheral and autonomic nervous tissues. Thus, other intrinsic factors may be involved in amyloid deposition in the nervous tissues of human FAP.     

A temporal and ultrastructural relationship between heparan sulfate proteoglycans and AA amyloid in experimental amyloidosis

Previous histochemical studies have suggested a close temporal relationship between the deposition of highly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA amyloidosis. In the present investigation, we extended these initial observations by using specific immunocytochemical probes to analyze the temporal and ultrastructural relationship between heparan sulfate proteoglycan (HSPG) accumulation and amyloid deposition in a mouse model of AA amyloidosis. Antibodies against the basement membrane-derived HSPG (either protein core or GAG chains) demonstrated a virtually concurrent deposition of HSPGs and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (amyloid enhancing factor + silver nitrate, or daily azocasein injections). Polyclonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to sites of HSPG deposition in amyloid sites, whereas no positive immunostaining was observed in these locales with a polyclonal antibody to the protein core of a dermatan sulfate proteoglycan (known as "decorin"). Immunogold labeling of HSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGs to amyloid fibrils. In the liver, heparan sulfate GAGs were also immunolocalized to the lysosomal compartment of hepatocytes and/or Kupffer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The close temporal and ultrastructural relationship between HSPGs and AA amyloid further implies an important role for HSPGs during the initial stages of AA amyloidosis.     

Role of the toll-like receptor 4 in neuroinflammation in Alzheimer's disease.

Microglial activation is a key feature in Alzheimer's disease and is considered to contribute to progressive neuronal injury by release of neurotoxic products. The innate immune receptor Toll-like-receptor 4 (TLR4), localized on the surface of microglia, is a first-line host defense receptor against invading microorganisms. Here, we show that a spontaneous loss-of-function mutation in the Tlr4 gene strongly inhibits microglial and monocytic activation by aggregated Alzheimer amyloid peptide resulting in a significantly lower release of the inflammatory products IL-6, TNFalpha and nitric oxide. Treatment of primary murine neuronal cells with supernatant of amyloid peptide-stimulated microglia demonstrates that Tlr4 contributes to amyloid peptide-induced microglial neurotoxicity. In addition, stimulation experiments in transfected HEK293 cells allowed to define a tri-molecular receptor complex consisting of TLR4, MD-2 and CD14 necessary for full cellular activation by aggregated amyloid peptide. A clinical relevance of these findings is supported by a marked upregulation of Tlr4 mRNA in APP transgenic mice and by an increased expression of TLR4 in Alzheimer's disease brain tissue associated with amyloid plaque deposition. Together, these observations provide the first evidence for a role of the key innate immune receptor, TLR4, in neuroinflammation in Alzheimer's disease.     

Amyloid β Protein (25–35) Stimulation of Phospholipase C in LA-N-2 Cells

Abstract: The amyloid β protein (25–35) stimulated appearance of ³H-inositol phosphates from [³H]inositol-prelabeled LA-N-2 cells was investigated. This stimulation was unaltered by extra- and intracellular calcium chelators in a calcium-free medium or by several protein kinase inhibitors. This phospholipase C stimulation by amyloid β protein appeared to be pertussis toxin sensitive. It is possible that this phospholipase C stimulation by amyloid β protein is a receptor-mediated process. This possibility is based on two related observations. The stimulation is ablated by the presence of conventional antagonists for metabotropic, adrenergic, and bombesin agonists. The IC₅₀ values were 12 µ M for propranolol, 15 µ M for AP-3, and 25 n M for [Tyr⁴, d -Phe¹²]bombesin. Additional support comes from results of densensitization and resensitization experiments. Amyloid β protein stimulation of phospholipase C was absent from LA-N-2 cells previously treated with norepinephrine, trans -1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), bombesin, or amyloid β peptide. In a similar manner, LA-N-2 cells previously treated with amyloid β protein were no longer responsive to norepinephrine, t-ACPD, or bombesin. The responsiveness to amyloid β protein returned, subsequent to a period of resensitization for the individual agonists. It is suggested that this observed amyloid β protein stimulation of phospholipase C may be responsible for the elevated quantity of inositol seen in the brains of Alzheimer's disease patients.     

Different adrenal sympathetic preganglionic neurons regulate epinephrine and norepinephrine secretion

Brain stimulation or activation of certain reflexes can result in differential activation of the two populations of adrenal medullary chromaffin cells: those secreting either epinephrine or norepinephrine, suggesting that they are controlled by different central sympathetic networks. In urethan-chloralose-anesthetized rats, we found that antidromically identified adrenal sympathetic preganglionic neurons (SPNs) were excited by stimulation of the rostral ventrolateral medulla (RVLM) with either a short (mean: 29 ms) or a long (mean: 129 ms) latency. The latter group of adrenal SPNs were remarkably insensitive to baroreceptor reflex activation but strongly activated by the glucopenic agent 2-deoxyglucose (2-DG), indicating their role in regulation of adrenal epinephrine release. In contrast, adrenal SPNs activated by RVLM stimulation at a short latency were completely inhibited by increases in arterial pressure or stimulation of the aortic depressor nerve, were unaffected by 2-DG administration, and are presumed to govern the discharge of adrenal norepinephrine-secreting chromaffin cells. These findings of a functionally distinct preganglionic innervation of epinephrine- and norepinephrine-releasing adrenal chromaffin cells provide a foundation for identifying the different sympathetic networks underlying the differential regulation of epinephrine and norepinephrine secretion from the adrenal medulla in response to physiological challenges and experimental stimuli.     

Specific pathogen free conditions prevent transthyretin amyloidosis in mouse models

Transthyretin (TTR) associated amyloidosis is an autosomal dominant disorder characterized by peripheral and autonomic neuropathy. Both genetic and environmental factors are thought to be involved in development of TTR associated amyloidosis. Previously, we demonstrated that amyloid deposition was observed in various tissues of transgenic mouse lines carrying a human mutant TTR (Met30) gene. To analyze the influence of environmental factors on TTR amyloidosis, these amyloidogenic transgenic mouse models were kept under conventional (CV) or specific pathogen free (SPF) conditions. Although the serum levels of Met30 for mice housed in the CV and SPF conditions were similar, amyloid deposition was observed in CV conditions, but not in SPF conditions. In addition, the extent of amyloid deposition in transgenic mice was dependent on duration kept under CV conditions. There were significant differences in proportion of amyloid deposition in several tissues between CV and SPF conditions. Maintenance of these mice at 30 degrees C did not induce amyloid deposition in SPF conditions. These results suggest that the SPF conditions can completely prevent amyloid deposition, and that environmental factors can affect the onset and progression even in a single gene disorder.     

Neprilysin Impedes Islet Amyloid Formation by Inhibition of Fibril Formation Rather Than Peptide Degradation

Deposition of islet amyloid polypeptide (IAPP) as islet amyloid in type 2 diabetes contributes to loss of β-cell function and mass, yet the mechanism for its occurrence is unclear. Neprilysin is a metallopeptidase known to degrade amyloid in Alzheimer disease. We previously demonstrated neprilysin to be present in pancreatic islets and now sought to determine whether it plays a role in degrading islet amyloid. We used an in vitro model where cultured human IAPP (hIAPP) transgenic mouse islets develop amyloid and thereby have increased β-cell apoptosis. Islet neprilysin activity was inhibited or up-regulated using a specific inhibitor or adenovirus encoding neprilysin, respectively. Following neprilysin inhibition, islet amyloid deposition and β-cell apoptosis increased by 54 and 75%, respectively, whereas when neprilysin was up-regulated islet amyloid deposition and β-cell apoptosis both decreased by 79%. To determine if neprilysin modulated amyloid deposition by cleaving hIAPP, analysis of hIAPP incubated with neprilysin was performed by mass spectrometry, which failed to demonstrate neprilysin-induced cleavage. Rather, neprilysin may act by reducing hIAPP fibrillogenesis, which we showed to be the case by fluorescence-based thioflavin T binding studies and electron microscopy. In summary, neprilysin decreases islet amyloid deposition by inhibiting hIAPP fibril formation, rather than degrading hIAPP. These findings suggest that targeting the role of neprilysin in IAPP fibril assembly, in addition to IAPP cleavage by other peptidases, may provide a novel approach to reduce and/or prevent islet amyloid deposition in type 2 diabetes.     

The release of catecholamines from the adrenal medulla and its modulation by alpha 2-adrenoceptors in the anaesthetized dog

The adrenal nerve of anaesthetized and vagotomized dogs was electrically stimulated (10 V pulses of 2 ms duration for 10 min) at frequencies of 1, 3, 10, and 25 Hz. There was a correlation between the frequency of stimulation and the plasma concentrations of epinephrine, norepinephrine, and dopamine in the adrenal vein, mainly after the 1st min of stimulation and the maximal concentration was reached sooner with higher frequencies of stimulation. Moreover, the relative percentage of catecholamines released in response to the electrical stimulation was not changed by the frequency of stimulation. To test the hypothesis that a local negative feedback mechanism mediated by alpha 2-adrenoceptors exists in the adrenal medulla, the effects of the systemic administration of clonidine (alpha 2-antagonist) on the concentrations of catecholamines in the adrenal vein were evaluated during the electrical stimulation of the adrenal nerve (5 V pulses of 2 ms duration for 3 min) at 3 Hz. Moreover, the effects of the systemic injections of more specific alpha 2-agonist and antagonist (oxymetazoline and idazoxan) were tested on the release of catecholamines in the adrenal vein in response to electrical stimulation of the splanchnic nerve at 1 and 3 Hz frequencies. The injection of 0.5 mg/kg of yohimbine caused a significant increase in the concentrations of epinephrine and norepinephrine in the adrenal vein induced by the electrical stimulation of the adrenal nerve and the injection of 15 micrograms/kg of clonidine had no effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular biology of the amyloid of Alzheimer's disease. An overview.

Alzheimer's disease is a progressive neurodegenerative disorder that affects a significant percentage of elderly individuals. Degenerative nerve cells express atypical proteins, and amyloid is deposited. The hallmark event of Alzheimer's disease is the deposition of amyloid as insoluble fibrous masses in extracellular neuritic plaques and around the walls of cerebral blood vessels. This review will focus on the advances on the knowledge of Alzheimer's amyloid, because it is becoming increasingly clear that the deposition of amyloid on neuritic plaques in the brain represents the earliest and most characteristic pathological feature of Alzheimer's disease. The main component of amyloid is a 4.2-4.5 KDa hydrophobic peptide, named amyloid beta-peptide, that is codified in chromosome 21 as part of a much larger precursor protein. The study of the mechanism by which the amyloid beta-peptide arises from the amyloid precursor protein is very important in order to understand the biological basis of amyloid deposition and its role in Alzheimer's disease.     

Comparison of amyloid deposition in two lines of transgenic mouse that model familial amyloidotic polyneuropathy,type I

We previously produced a transgenic mouse line designated MT-hMet30 by introducing the human mutant transthyretin (TTR) gene carrying the mouse metallothionein promoter, and showed that the presence of human variant TTR is sufficient for amyloid deposition in various tissues of these transgenic mice. However, the expression pattern of human mutant transthyretin gene in the mouse was different from that in man. To analyse pathologic processes, it is essential to establish a transgenic mouse line in which the developmental and tissue- specific expression of the human mutant TTR gene is the same as in man. Thus, we produced two additional transgenic mouse lines carrying the human mutant TTR gene containing either 0.6 kb (0.6- hMet30) or 6.0 kb (6.0-hMet30) of the upstream region. The expression levels of 6.0-hMet30 gene in the liver and serum were the same as in man and about 10 times higher than those of 0.6- hMet30 gene. In both lines amyloid deposition was observed in similar tissues to human patients except for the peripheral and autonomic nervous tissues. The amyloid deposition started earlier and was more extensive in 6.0-hMet30 than 0.6-hMet30 mice, suggesting that the serum levels of human mutant TTR are correlated with the occurrence and degree of amyloid deposition, to some extent. Neither amyloid deposition nor degenerative changes were observed in the peripheral and autonomic nervous systems despite the transgene expression in the choroid plexus of the 6.0-hMet30 mice. In the 6.0-hMet30 mice, amyloid deposition started at 9 months of age, although the serum level of human mutant TTR reached the adult level at 1 month. These results suggest that intrinsic environmental factors other than the mutant gene are involved in the late-onset deposition of amyloid fibrils. Transgenic mice described here should be useful for analysing such factors     

Longitudinal ultrastructure study of islet amyloid in the HIP rat model of type 2 diabetes mellitus

In 2004, the human islet amyloid polypeptide (HIP) rat model was created by transfecting the Sprague-Dawley rat with the human islet amyloid polypeptide (hIAPP)-amylin gene. The objective of this study is to utilize the transmission electron microscope to study the longitudinal cellular and extracellular morphological changes within the islets of this model at 4, 8, and 14 months of age. It has been previously demonstrated that the 2-, 5-, and 10-month HIP models have no diabetes, impaired fasting glucose, and diabetes, respectively. The 4-month HIP model (FBS 123 mg/dl) demonstrated an abundance of beta-cells and insulin secretory granules with significant pericapillary and inter-beta-cell islet amyloid deposition. The 8-month model (FBS 187 mg/dl) demonstrated extensive islet amyloid deposition and marked changes of beta-cell apoptosis. The 14-month-old model (FBS 244 mg/dl) demonstrated islet and beta-cell atrophy with even greater amounts of extracellular islet amyloid compared to the 4-month-old and 8-month-old models. Functional beta cells were sparse and were associated with intra islet adipose deposition. These findings of ultrastructure cellular and extracellular morphological longitudinal remodeling changes in this novel animal model of type 2 diabetes may provide investigators with a better understanding regarding the role of islet amyloid in human islet.     

Electron microscopic study on amyloid fibril formation in human lymph nodes

The purpose of this investigation was to clarify the mechanisms of amyloid fibril formation in human lymph nodes. In our present study, amyloid deposition was observed diffusely in all compartments of the lymph nodes. The deposition form showed extremely characteristic findings in its morphological features. Namely, amyloid deposits mainly consisted of clusters of round or oval nodules. Each amyloid nodule was frequently enclosed with long-stretched cytoplasmic processes of abutting reticulum cells and/or macrophages. Amyloid fibrils often formed parallel amyloid bundles radiating to outlying sections of the nodule from the center. The amyloid bundles closely adhered to the cytoplasmic membrane of not only the abutting reticulum cells, macrophages and sinus endothelium but also to the lymphocytes and plasma cells. In the central portion of the amyloid nodules, a concentric core was also observed. The most interesting finding was the intracellular formation of amyloid fibrils in all cells, such as macrophages, reticulum cells, foreign body giant cells and lymphocytes in the process of degeneration. Some fibrils localized in the limited area of the cytoplasm and others appeared in all parts of the cells, including the nucleus. Their cell membranes were missing in several areas and the cell organella had gradually dissolved. Finally the cell residuums were completely replaced by amyloid fibrils and transformed into a nodular structure with radiating bundles of amyloid fibrils.     

Matrix Metalloproteinase-9 Reduces Islet Amyloid Formation by Degrading Islet Amyloid Polypeptide

Deposition of islet amyloid polypeptide (IAPP) as amyloid is a pathological hallmark of the islet in type 2 diabetes, which is toxic to β-cells. We previously showed that the enzyme neprilysin reduces islet amyloid deposition and thereby reduces β-cell apoptosis, by inhibiting fibril formation. Two other enzymes, matrix metalloproteinase (MMP)-2 and MMP-9, are extracellular gelatinases capable of degrading another amyloidogenic peptide, Aβ, the constituent of amyloid deposits in Alzheimer disease. We therefore investigated whether MMP-2 and MMP-9 play a role in reducing islet amyloid deposition. MMP-2 and MMP-9 mRNA were present in mouse islets but only MMP-9 activity was detectable. In an islet culture model where human IAPP (hIAPP) transgenic mouse islets develop amyloid but nontransgenic islets do not, a broad spectrum MMP inhibitor (GM6001) and an MMP-2/9 inhibitor increased amyloid formation and the resultant β-cell apoptosis. In contrast, a specific MMP-2 inhibitor had no effect on either amyloid deposition or β-cell apoptosis. Mass spectrometry demonstrated that MMP-9 degraded amyloidogenic hIAPP but not nonamyloidogenic mouse IAPP. Thus, MMP-9 constitutes an endogenous islet protease that limits islet amyloid deposition and its toxic effects via degradation of hIAPP. Because islet MMP-9 mRNA levels are decreased in type 2 diabetic subjects, islet MMP-9 activity may also be decreased in human type 2 diabetes, thereby contributing to increased islet amyloid deposition and β-cell loss. Approaches to increase islet MMP-9 activity could reduce or prevent amyloid deposition and its toxic effects in type 2 diabetes.     

Amyloid-enhancing factor (AEF) in the pathogenesis of AA-amyloidosis in the hamster

AA-amyloidosis was induced in hamsters receiving amyloid-enhancing factor (AEF) by daily subcutaneous injection with either an aged casein solution or casein supplemented with lipopolysaccharide (LPS). Both amyloid inducers gave similar results with respect to amyloid development in spleen, liver and kidneys and to serum amyloid A (SAA) concentrations and plasma cathepsin D activities. AEF was isolated from amyloid-containing tissue by the method described by Hol et al. (1985), and amyloid-enhancing material was also extracted from isolated hamster amyloid fibrils by intensive sonification. This fibril-derived amyloid-enhancing material lacked typical green birefringence after staining with Congo red and appeared as amorphous material on electron microscopy. AEF shortened the pre-amyloid phase for splenic and hepatic amyloid development and also the subsequent interval before renal amyloid deposition. This indicates that endogenous AEF, unlike passively transferred preformed AEF, is not distributed throughout the body and is probably generated at the site of amyloid deposition. Moreover, these results suggest that amyloid deposition in the kidneys, like that in the spleen and liver, involves an AEF-dependent pathway. Thus redistribution of amyloid is probably not an important cause of renal amyloid involvement. In addition to the reduction in the lag phase for splenic and hepatic amyloid deposition, AEF also speeds the changes in SAA concentration and plasma cathepsin D activity. This indicates that AEF accelerates rather than eliminates the pre-amyloid phase.     

Metal ions differentially influence the aggregation and deposition of Alzheimer's beta-amyloid on a solid template

The abnormal deposition and aggregation of beta-amyloid (Abeta) on brain tissues are considered to be one of the characteristic neuropathological features of Alzheimer's disease (AD). Environmental conditions such as metal ions, pH, and cell membranes are associated with Abeta deposition and plaque formation. According to the amyloid cascade hypothesis of AD, the deposition of Abeta42 oligomers as diffuse plaques in vivo is an important earliest event, leading to the formation of fibrillar amyloid plaques by the further accumulation of soluble Abeta under certain environmental conditions. In order to characterize the effect of metal ions on amyloid deposition and plaque growth on a solid surface, we prepared a synthetic template by immobilizing Abeta oligomers onto a N-hydroxysuccinimide ester-activated solid surface. According to our study using ex situ atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), and thioflavin T (ThT) fluorescence spectroscopy, Cu2+ and Zn2+ ions accelerated both Abeta40 and Abeta42 deposition but resulted only in the formation of "amorphous" aggregates. In contrast, Fe3+ induced the deposition of "fibrillar" amyloid plaques at neutral pH. Under mildly acidic environments, the formation of fibrillar amyloid plaques was not induced by any metal ion tested in this work. Using secondary ion mass spectroscopy (SIMS) analysis, we found that binding Cu ions to Abeta deposits on a solid template occurred by the possible reduction of Cu ions during the interaction of Abeta with Cu2+. Our results may provide insights into the role of metal ions on the formation of fibrillar or amorphous amyloid plaques in AD.     

Simultaneous monitoring of acetylcholine and catecholamine release in the in vivo rat adrenal medulla

To simultaneously monitor acetylcholine release from pre-ganglionic adrenal sympathetic nerve endings and catecholamine release from post-ganglionic adrenal chromaffin cells in the in vivo state, we applied microdialysis technique to anesthetized rats. Dialysis probe was implanted in the left adrenal medulla and perfused with Ringer's solution containing neostigmine (a cholinesterase inhibitor). After transection of splanchnic nerves, we electrically stimulated splanchnic nerves or locally administered acetylcholine through dialysis probes for 2 min and investigated dialysate acetylcholine, choline, norepinephrine and epinephrine responses. Acetylcholine was not detected in dialysate before nerve stimulation, but substantial acetylcholine was detected by nerve stimulation. In contrast, choline was detected in dialysate before stimulation, and dialysate choline concentration did not change with repetitive nerve stimulation. The estimated interstitial acetylcholine levels and dialysate catecholamine responses were almost identical between exogenous acetylcholine (10 microM) and nerve stimulation (2 Hz). Dialysate acetylcholine, norepinephrine and epinephrine responses were correlated with the frequencies of electrical nerve stimulation, and dialysate norepinephrine and epinephrine responses were quantitatively correlated with dialysate acetylcholine responses. Neither hexamethonium (a nicotinic receptor antagonist) nor atropine (a muscarinic receptor antagonist) affected the dialysate acetylcholine response to nerve stimulation. Microdialysis technique made it possible to simultaneously assess activities of pre-ganglionic adrenal sympathetic nerves and post-ganglionic adrenal chromaffin cells in the in vivo state and provided quantitative information about input-output relationship in the adrenal medulla.