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Abnormal function of potassium channels in platelets of patients with Alzheimer's disease

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Abstract

Reports of abnormalities of potassium-channel function in various cultured cells of Alzheimer's disease patients led us to attempt to characterise the pharmacological characteristics of the abnormal channel. We studied platelets from 14 patients with Alzheimer-type dementia and 14 non-demented controls matched for age and sex. The effects of specific inhibitors of K+ channels on the efflux of rubidium-86 ions, a radioactive analogue of K+, from the platelets were measured. Normal platelets contain three types of K+ channel, sensitive to the inhibitory actions of apamin (small-conductance calcium-dependent potassium channels), charybdotoxin (of less specificity, but probably intermediate-conductance calcium-dependent K+ channels), and alpha-dendrotoxin (voltage-sensitive K+ channels). However, 8Rb+ efflux from the platelets of patients with Alzheimer-type dementia was not inhibited by either apamin or charybdotoxin. By contrast, inhibition by alpha-dendrotoxin did occur. Our results suggest that calcium-dependent K+ channels in platelets are selectively impaired in Alzheimer's disease. A similar abnormality in neurons could contribute to the pathophysiology of the disorder.

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... Although initially differentiated on the basis of biophysical and differential toxin sensitivity, distinct genes are now known to encode various calcium-activated K channels (Vergara et al., 1998;Castle, 1999;Wallner et al., 1999b). Abnormal function of calcium-activated K channels has been noted in platelets of patients with Alzheimer's disease, although its relevance to the pathology is not clear (de Silva et al., 1998). The CAG triplet repeat in KCNN3 gene encoding a small conductance calciumactivated K channel, hKCa3, mapped to chromosome 1q21 has been reported to be associated with schizophrenia ( Chandy et al., 1998), although subsequent investigations to confirm these findings have been met with mixed results ( Austin et al., 1999;Dror et al., 1999). ...
... TEA depolarized and elevated intracellular Ca 2 levels in young and aged control fibroblasts but not in fibroblasts from Alzheimer's disease patients, supporting the dysfunction of TEA-sensitive K channels in the disease. Rb flux through apamin and charybdotoxin-sensitive Ca 2-activated K channels was selectively impaired in fresh, noncultured platelets from patients with Alzheimer's-type dementia, although the-dendrotoxin-sensitive voltage-dependent K channel was not affected compared with nondemented controls (de Silva et al., 1998).-Amyloid protein also enhanced phytohemagglutinin-induced Ca 2 rise in T-lymphocytes, consistent with the hypothesis that enhanced calcium responses serve as a general feature of-amyloid neurotoxicity ( Eckert et al., 1993). ...
Article
Potassium channels play important roles in vital cellular signaling processes in both excitable and nonexcitable cells. Over 50 human genes encoding various K(+) channels have been cloned during the past decade, and precise biophysical properties, subunit stoichiometry, channel assembly, and modulation by second messenger and ligands have been elucidated to a large extent. Recent advances in genetic linkage analysis have greatly facilitated the identification of many disease-producing loci, and naturally occurring mutations in various K(+) channels have been identified in diseases such as long-QT syndromes, episodic ataxia/myokymia, familial convulsions, hearing and vestibular diseases, Bartter's syndrome, and familial persistent hyperinsulinemic hypoglycemia of infancy. In addition, changes in K(+) channel function have been associated with cardiac hypertrophy and failure, apoptosis and oncogenesis, and various neurodegenerative and neuromuscular disorders. This review aims to 1) provide an understanding of K(+) channel function at the molecular level in the context of disease processes and 2) discuss the progress, hurdles, challenges, and opportunities in the exploitation of K(+) channels as therapeutic targets by pharmacological and emerging genetic approaches.
... There are several reports of the effect of scorpion venom on platelets. Charybdotoxin is a toxin in Leiurus quinquestriatus venom that inhibits the procoagulant response of human platelets through Ca 2þactivated potassium channels [5][6][7]. A bioactive polypeptide from Buthus martensii Karsh venom has been reported to inhibit rabbit and rat platelet aggregation by thrombin or ADP but the underlying mechanism is not known [8]. ...
... There are several reports of the effect of scorpion venom on platelets channels. However, the majority of these toxins inhibit the procoagulant response of human platelets through Ca 2þ -activated potassium channels [5][6][7] and at the present time there are no reports that the scorpion toxins that have an effect on platelets channels promote aggregation. The low molecular weight proteins in TdV have been characterized using proteomics [18]. ...
Article
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In humans and other mammals, Tityus discrepans (Td) scorpion envenomation produces a variety of systemic effects including respiratory distress, a generalized inflammatory reaction, modulation of blood pressure, fibrin formation, and platelet activation. For many of these effects, the venom components and underlying mechanisms are not known. In the present study, we demonstrate that Td venom (TdV) stimulates integrin αIIbβ3-dependent aggregation of washed human and mouse platelets downstream of Src kinase activation. The pattern of increase in tyrosine phosphorylation induced by TdV in human platelets is similar to that induced by the collagen receptor GPVI, and includes FcR γ-chain, Syk, and PLC γ 2. Confirmation of GPVI activation by TdV was achieved by expression of human GPVI in chicken DT40 B cells and use of a reporter assay. To our surprise, TdV was able to activate mouse platelets deficient in the GPVI-FcR γ-chain complex through a pathway that was also dependent on Src kinases. TdV therefore activates platelets through GPVI and a second, as yet unidentified Src kinase-dependent pathway.
... Although experimental approaches indicate that A deposition plays an important role in the neurodegenerative process in AD [7,8], the mechanism underlying the relentless progression of neurofibrillary pathology and neuronal death remains to be fully elucidated. Over the last decade there have been a number of studies demonstrating that potassium channel dysfunction may be involved in the pathogenesis of AD [9][10][11][12][13]. ...
... The Kv4 channels are composed of the pore-forming subunits, various auxiliary subunits, and other interacting proteins such as Kv channel interacting proteins and dipeptidyl peptidase-like proteins DPP6 and DPP10 [15][16][17]. Indeed, potassium channel dysfunction has been demonstrated in fibroblasts and platelets of AD patients [9,10]. Postmortem studies have also showed alterations of potassium channel expression in AD brains [18]. ...
Article
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The neuropathological features associated with Alzheimer’s disease (AD) include the presence of extracellular amyloid-β peptide-containing plaques and intracellular tau positive neurofibrillary tangles and the loss of synapses and neurons in defined regions of the brain. Dipeptidyl peptidase 10 (DPP10) is a protein that facilitates Kv4 channel surface expression and neuronal excitability. This study aims to explore DPP10789 protein distribution in human brains and its contribution to the neurofibrillary pathology of AD and other tauopathies. Immunohistochemical analysis revealed predominant neuronal staining of DPP10789 in control brains, and the CA1 region of the hippocampus contained strong reactivity in the distal dendrites of the pyramidal cells. In AD brains, robust DPP10789 reactivity was detected in neurofibrillary tangles and plaque-associated dystrophic neurites, most of which colocalized with the doubly phosphorylated Ser-202/Thr-205 tau epitope. DPP10789 positive neurofibrillary tangles and plaque-associated dystrophic neurites also appeared in other neurodegenerative diseases such as frontotemporal lobar degeneration, diffuse Lewy body disease, and progressive supranuclear palsy. Occasional DPP10789 positive neurofibrillary tangles and neurites were seen in some aged control brains. Western blot analysis showed both full length and truncated DPP10789 fragments with the later increasing significantly in AD brains compared to control brains. Our results suggest that DPP10789 is involved in the pathology of AD and other neurodegenerative diseases.
... Although experimental approaches indicate that A deposition plays an important role in the neurodegenerative process in AD [7,8], the mechanism underlying the relentless progression of neurofibrillary pathology and neuronal death remains to be fully elucidated. Over the last decade there have been a number of studies demonstrating that potassium channel dysfunction may be involved in the pathogenesis of AD [9][10][11][12][13]. ...
... The Kv4 channels are composed of the pore-forming subunits, various auxiliary subunits, and other interacting proteins such as Kv channel interacting proteins and dipeptidyl peptidase-like proteins DPP6 and DPP10 [15][16][17]. Indeed, potassium channel dysfunction has been demonstrated in fibroblasts and platelets of AD patients [9,10]. Postmortem studies have also showed alterations of potassium channel expression in AD brains [18]. ...
Article
Full-text available
The neuropathological features associated with Alzheimer's disease (AD) include the presence of extracellular amyloid-β peptide-containing plaques and intracellular tau positive neurofibrillary tangles and the loss of synapses and neurons in defined regions of the brain. Dipeptidyl peptidase 10 (DPP10) is a protein that facilitates Kv4 channel surface expression and neuronal excitability. This study aims to explore DPP10789 protein distribution in human brains and its contribution to the neurofibrillary pathology of AD and other tauopathies. Immunohistochemical analysis revealed predominant neuronal staining of DPP10789 in control brains, and the CA1 region of the hippocampus contained strong reactivity in the distal dendrites of the pyramidal cells. In AD brains, robust DPP10789 reactivity was detected in neurofibrillary tangles and plaque-associated dystrophic neurites, most of which colocalized with the doubly phosphorylated Ser-202/Thr-205 tau epitope. DPP10789 positive neurofibrillary tangles and plaque-associated dystrophic neurites also appeared in other neurodegenerative diseases such as frontotemporal lobar degeneration, diffuse Lewy body disease, and progressive supranuclear palsy. Occasional DPP10789 positive neurofibrillary tangles and neurites were seen in some aged control brains. Western blot analysis showed both full length and truncated DPP10789 fragments with the later increasing significantly in AD brains compared to control brains. Our results suggest that DPP10789 is involved in the pathology of AD and other neurodegenerative diseases.
... Platelet function is disrupted in AD, and platelet activation status is thought to be a biomarker for disease progression [274,275]. In contrast, a recent study of platelet migration into the brain parenchyma in an AD mouse model found just a few platelets moving into the brain parenchyma [276]. ...
Article
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Platelets play a variety of roles in vascular biology and are best recognized as primary hemostasis and thrombosis mediators. Platelets have a large number of receptors and secretory molecules that are required for platelet functionality. Upon activation, platelets release multiple substances that have the ability to influence both physiological and pathophysiological processes including inflammation, tissue regeneration and repair, cancer progression, and spreading. The involvement of platelets in the progression and seriousness of a variety of disorders other than thrombosis is still being discovered, especially in the areas of inflammation and the immunological response. This review represents an integrated summary of recent advances on the function of platelets in pathophysiology that connects hemostasis, inflammation, and immunological response in health and disease and suggests that antiplatelet treatment might be used for more than only thrombosis.
... In fact, oxidative stress is a well-established cause of AD preceding and later stimulating the formation of b-amyloid plaque (reviewed in Ref. [2]). This in turn increases the net surplus of ROS [50,51] in a type of autocatalytic process (bamyloid clogs the mitochondrial protein import machinery, causing mitochondrial dysfunction and impaired energy metabolism, inhibiting cytochrome oxidase activity, and thus increasing free-radical generation). The increased availability of ROS in mouse models of AD could therefore explain why the BK channels are more active – they are more extensively oxidized than in normal mice. ...
Article
A wealth of evidence underscores the tight link between oxidative stress, neurodegeneration and aging. When the level of excess reactive oxygen species (ROS) increases in the cell, a phenomenon characteristic of aging, DNA is damaged, proteins are oxidized, lipids are degraded and more ROS are produced, all culminating in significant cell injury. Recently we showed that in the nematode, Caenorhabditis elegans, oxidation of K(+) channels by ROS is a major mechanism underlying the loss of neuronal function. The C. elegans results support an argument that K(+) channels controlling neuronal excitability and survival might provide a common, functionally important substrate for ROS in aging mammals. Here we discuss the implications that oxidation of K(+) channels by ROS might have for the mammalian brain during normal aging, as well as in neurodegenerative diseases such as Alzheimer's and Parkinson's. We argue that oxidation of K(+) channels by ROS is a common theme in the aging brain and suggest directions for future experimentation.
... Alterations to platelet Ca 2+ -dependent K + channel function have been suggested to occur in Alzheimer's disease [86]. This neurodegenerative disease is characterized pathologically by the appearance of proteinaceous plaques in areas of the brain [87]. ...
Article
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Potassium ions have widespread roles in cellular homeostasis and activation as a consequence of their large outward concentration gradient across the surface membrane and ability to rapidly move through K⁺-selective ion channels. In platelets, the predominant K⁺ channels include the voltage-gated K⁺ channel Kv1.3, and the intermediate conductance Ca²⁺-activated K⁺ channel KCa3.1, also known as the Gardos channel. Inwardly rectifying potassium GIRK channels and KCa1.1 large conductance Ca²⁺-activated K⁺ channels have also been reported in the platelet, although they remain to be demonstrated using electrophysiological techniques. Whole-cell patch clamp and fluorescent indicator measurements in the platelet or their precursor cell reveal that Kv1.3 sets the resting membrane potential and KCa3.1 can further hyperpolarize the cell during activation, thereby controlling Ca²⁺ influx. Kv1.3-/- mice exhibit an increased platelet count, which may result from an increased splenic megakaryocyte development and longer platelet lifespan. This review discusses the evidence in the literature that Kv1.3, KCa3.1. GIRK and KCa1.1 channels contribute to a number of platelet functional responses, particularly collagen-evoked adhesion, procoagulant activity and GPCR function. Putative roles for other K⁺ channels and known accessory proteins which to date have only been detected in transcriptomic or proteomic studies, are also discussed.
... On the other hand, potassium channel abnormalities have been reported in both neural and peripheral tissues of AD patients. In particular, K + channel dysfunction has been demonstrated in fibroblasts [36] and platelets [37] of AD patients and post-mortem studies showed alterations of K + channel expression in the brain [38,39]. Moreover, an aberrant glutamate-dependent modulation of Kv1.3 channels was recently demonstrated in T lymphocytes from AD patients [40]. ...
Article
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Reduced levels of Substance P (SP), an endogenous neuropeptide endowed with neuroprotective and anti-apoptotic properties, have been found in brain and spinal fluid of Alzheimer's disease (AD) patients. Potassium (K+) channel dysfunction is implicated in AD development and the amyloid-β (Aβ)-induced up-regulation of voltage-gated potassium channel subunits could be considered a significant step in Aβ brain toxicity. The aim of this study was to evaluate whether SP could reduce, in vivo, Aβ-induced overexpression of Kv subunits. Rats were intracerebroventricularly infused with amyloid-β 25–35 (Aβ25–35, 20 µg) peptide. SP (50 µg/Kg, i.p.) was daily administered, for 7 days starting from the day of the surgery. Here we demonstrate that the Aβ infused rats showed impairment in cognitive performances in the Morris water maze task 4 weeks after Aβ25–35 infusion and that this impairing effect was prevented by SP administration. Kv1.4, Kv2.1 and Kv4.2 subunit levels were quantified in hippocampus and in cerebral cortex by Western blot analysis and immunofluorescence. Interestingly, SP reduced Kv1.4 levels overexpressed by Aβ, both in hippocampus and cerebral cortex. Our findings provide in vivo evidence for a neuroprotective activity of systemic administration of SP in a rat model of AD and suggest a possible mechanism underlying this effect.
... Many factors are responsible for Alzheimer. Some researchers have suggested that it can be due to calcium-dependent K+ channels in platelets [3] while Lanne has given the concept the ryanodine receptors (RyRs) which is the major intracellular Ca 2+ release channel [4] as a probable cause. However diets (proteins) [5], [6] and hormones may also play a pivotal role [7]- [10]. ...
... 5,7 In addition, we and others have observed that platelet hyperpolarization has the potential to directly reduce platelet activation. 5,8,9 However, it remains to be shown, whether there is a continuous release of EDHF in vivo and whether in view of the diluting effect of streaming blood the arteriolar endothelium releases sufficient amounts of EDHF to inhibit platelet activity as assessed by platelet interaction with the vessel wall in vivo. If so, this factor could be of significant importance not only for the endogenous protection from thrombotic disease but also atherosclerosis since activation of platelets contributes to the development of cardiovascular disorders. ...
Article
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A CYP2C9-dependent endothelium-derived hyperpolarizing factor (EDHF) controls blood flow in many microvascular beds of various species by targeting vascular smooth muscle potassium channels. Since platelets express the same channels, we tested whether EDHF hyperpolarizes platelets and exerts an antithrombotic function in vivo. Interaction of injected human platelets with the arteriolar wall (platelet-vessel wall interaction, PVWI) was assessed by intravital microscopy in skin muscle of awake hamsters. To understand the mechanisms of EDHF-induced platelet inhibition, we studied whether cultured human umbilical vein endothelial cells overexpressing CYP2C9-mRNA in vitro released a factor that could hyperpolarize human platelets. Under control conditions, there was no firm adhesion of platelets to the arteriolar wall, but temporary PVWI occurred. Local superfusion of the CYP2C9 inhibitor sulfaphenazole, at doses known to block EDHF-dependent dilations, significantly augmented PVWI, as did inhibition of NO synthase. Inhibition of both factors exerted additive effects on PVWI. Likewise, firm adhesion of a small fraction of platelets was observed. The prothrombotic effects of CYP2C9 inhibition in vivo were reversed by exogenous superfusion with 11,12-epoxyeicosatrienoic acids. Hyperpolarization reduced platelet adhesion to endothelial cells under static conditions in vitro and was dependent on calcium-activated potassium channels. The factor also reduced ADP-induced expression of platelet P-selectin, indicating reduction of platelet activity. The arteriolar endothelium in vivo continuously releases a CYP2C9-derived EDHF. This EDHF exerts its effects by hyperpolarization of platelets through activation of K(Ca) channels and reduction of platelet adhesion molecule expression, indicating that hyperpolarization reduces platelet activation. This demonstrates that EDHF is part of the antithrombotic properties of healthy endothelium in vivo.
... Modifications of potassium channel gating and permeation properties are thus likely to have a major impact on the mitochondria-dependent cellular functions. Notably, potassium channel dysfunctions have already been documented in fibroblasts (Etcheberrigaray et al., 1993) and platelets (de Silva et al., 1998) from AD patients, and post mortem studies, with their inherent limitations, have also showed alterations of potassium channel expression in AD brains (Ikeda et al., 1991). Relevant to the present study is the observation that peptide products of the amyloid precursor protein (APP), crucial in AD pathogenesis, could affect potassium channel activity (Plant et al., 2006) as well as components of intracellular pathways known to be involved in potassium channel modulation (Parameshwaran et al., 2008). ...
... Indeed, K + channel dysfunction has been demonstrated in fibroblasts (Etcheberrigaray et al., 1993) and platelets (De Silva et al., 1998) of AD patients. Postmortem studies, with their inherent limitations, also show alterations of K + channel expression in AD brains (Ikeda et al., 1991). ...
Article
Full-text available
In Alzheimer's disease (AD), potassium channel abnormalities have been reported in both neural and peripheral tissues. Herein, using whole-cell patch-clamp, we demonstrate an aberrant glutamate-dependent modulation of K(V)1.3 channels in T lymphocytes of AD patients. Although intrinsic K(V)1.3 properties in patients were similar to healthy individuals, glutamate (1-1000 microM) failed to yield the hyperpolarizing shift normally observed in K(V)1.3 steady-state inactivation (-4.4+/-2.7 mV in AD vs. -14.3+/-2.5 mV in controls, 10 microM glutamate), resulting in a 4-fold increase of resting channel activity. Specific agonist and antagonist data indicate that this abnormality is due to dysfunction of cognate group II mGluRs. Given that glutamate is present in plasma and that both mGluRs and K(V)1.3 channels regulate T-lymphocyte responsiveness, our finding may account for the presence of immune-associated alterations in AD. Furthermore, if this aberration reflects a corresponding one in neural tissue, it could provide a potential target in AD pathogenesis.
... Regarding SKCa channels in other cell types next to neurons in AD, it has been found that even if they are present in platelets, they were unresponsive to their inhibition (de Silva et al. 1998). SKCa channels play also a role in aging-related endothelial dysfunction, which is in part manifested as an impairment of NOmediated relaxation, but interestingly a reduced function of SKCa channels was also found . ...
Article
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Population aging, as well as the handling of age-associated diseases, is a worldwide increasing concern. Among them, Alzheimer's disease stands out as the major cause of dementia culminating in full dependence on other people for basic functions. However, despite numerous efforts, in the last decades, there was no new approved therapeutic drug for the treatment of the disease. Calcium-activated potassium channels have emerged as a potential tool for neuronal protection by modulating intracellular calcium signaling. Their subcellular localization is determinant of their functional effects. When located on the plasma membrane of neuronal cells, they can modulate synaptic function, while their activation at the inner mitochondrial membrane has a neuroprotective potential via the attenuation of mitochondrial reactive oxygen species in conditions of oxidative stress. Here we review the dual role of these channels in the aging phenotype and Alzheimer's disease pathology and discuss their potential use as a therapeutic tool.
... This property leads to neurotoxicity reflected in increased muscle activity, tremors, and fasciculations of the prey. Some structural analogs of dendrotoxin have led to the molecular recognition of different types of potassium channels, whose clinical utility is related to the study and possible development of treatments for demyelinating or neurodegenerative disorders [109,110]. ...
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Snake venoms have components with diverse biological actions that are extensively studied to identify elements that may be useful in biomedical sciences. In the field of autoimmunity and rheumatology, various findings useful for the study of diseases and potential drug development have been reported. The study of disintegrins, proteins that block the action of integrins, has been useful for the development of antiplatelet agents and principles for the development of immunosuppressants and antineoplastics. Several proteins in snake venoms act on the coagulation cascade, activating factors that have allowed the development of tests for the study of coagulation, including Russell’s viper venom time, which is useful in the diagnosis of antiphospholipid syndrome. Neurotoxins with either pre- or postsynaptic effects have been used to study neurogenic synapses and neuromuscular plaques and the development of analgesics, muscle relaxants and drugs for neurodegenerative diseases. Various components act by inhibiting cells and proteins of the immune system, which will allow the development of anti-inflammatory and immunosuppressive drugs. This review summarizes the usefulness of the components of snake venoms in the fields of autoimmunity and rheumatology, which can serve as a basis for diverse translational research.
... However, the absence of clear indices of epileptiform abnormalities is not a critical violation of the channelopathy criteria. The notion of channelopathy may well be expanded into the territory of neurodegenerative disorders , such as Alzheimer's disease, [59,103] which is infrequently associated with epileptiform phenomena. On the other hand, neurologic AN complications comprise the majority of those consistent with the presence of channelopathies: neuromuscular abnormalities (45%); generalized muscle weakness (43%); peripheral neuropathies (13%); headaches (6%); syncope (4%); diplopia (4%), and movement disorders (2%). ...
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Anorexia nervosa is a clinically significant illness that may be associated with permanent medical complications involving almost every organ system. The paper raises a question whether some of them are associated with premorbid vulnerability such as subcellular ion channel abnormalities ('channelopathy') that determines the clinical expression of the bodily response to self-imposed malnutrition. Aberrant channels emerge as a tempting, if rather speculative alternative to the notion of cognitively-driven neurotransmitter modulation deficit in anorexia nervosa. The concept of channelopathies is in keeping with some characteristics of anorexia nervosa, such as a genetically-based predisposition to hypophagia, early onset, cardiac abnormalities, an appetite-enhancing efficacy of some antiepileptic drugs, and others. The purpose of this article is to stimulate further basic research of ion channel biophysics in relation to restrictive anorexia.
... These, presumably, underlie their capacity for intimate cross-talk with inflammatory cells (5). To date, platelets have been implicated in the etiology of infection (6), inflammatory diseases including rheumatoid arthritis (7) and atherosclerosis (8), as well as neuropathologies, particularly stroke (9,10), Alzheimer disease (11)(12)(13) and, more recently, multiple sclerosis (MS) (14). ...
Article
Despite growing evidence for platelets as active players in infection and immunity, it remains unresolved whether platelets contribute to, or are key elements in the development of neuroinflammation. Using the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, we identified platelet accumulation in the circulation by 7-day postinduction (dpi), ahead of clinical onset which occurs at 13–14 dpi. By inducing platelet depletion between 7 and 16 dpi, we demonstrate an association between platelet accumulation in the spinal cord and disease development. Additionally, we provide evidence for platelet infiltration in the white and gray matter parenchyma, but with different outcomes. Thus, while in white matter platelets are clearly associated with lesions, in gray matter large-scale platelet infiltration and expression of the platelet-specific molecule PF4 are detectable prior to T cell entry. In the retina, platelet accumulation also precedes clinical onset and is associated with significant increase in retinal thickness in experimental relative to control animals. Platelet accumulation increases over the disease course in this tissue, but without subsequent T cell infiltration. These findings provide definitive confirmation that platelet accumulation is key to EAE pathophysiology. Furthermore, they suggest an undescribed and, most importantly, therapeutically targetable mechanism of neuronal damage.
... It appears that K + channels are naturally subjected to oxidation by ROS in both aging and neurodegenerative conditions characterized by high levels of ROS (Sesti, 2016). A dysfunction of K + channels has been observed in fibroblasts (Etcheberrigaray et al., 1993) and platelets (de Silva et al., 1998) of AD patients. Post-mortem studies have also reported an alteration in K + channels expression in AD brains (Ikeda et al., 1991). ...
Article
With more than 80 subunits, potassium (K+) channels represent a group of ion channels showing high degree of diversity and ubiquity. They play important role in the control of membrane depolarization and cell excitability in several tissues, including the brain. Controlling the intracellular and extracellular K+ flow in cells, they also modulate the hormone and neurotransmitter release, apoptosis and cell proliferation. It is therefore not surprising that an improper functioning of K+ channels in neurons has been associated with pathophysiology of a wide range of neurological disorders, especially Alzheimer’s disease (AD). This review aims to give a comprehensive overview of the basic properties and pathophysiological functions of the main classes of K+ channels in the context of disease processes, also discussing the progress, challenges and opportunities to develop drugs targeting these channels as potential pharmacological approach for AD treatment.
... Ultrastructural studies have revealed an increased proliferation of internal membranes, 15 while biophysical studies demonstrated a higher fluidity of intracellular membranes, but no change in plasma membrane fluidity in platelets of AD patients as compared to control subjects. 16 Calcium-dependent K + channels (voltagesensitive) of platelets are selectively impaired in AD. 17 A similar abnormality has been reported in AD hippocampus and could contribute to the pathophysiology of the disorder. Recently, increased levels of A␤, increased activation of ␤-secretase (BACE1), decreased activation of ␣-secretase (ADAM10) and decreased APP ratios have been found in AD platelets compared to normal control subjects, 5 suggesting that platelets can be considered a useful marker for the diagnosis of AD. ...
Article
The main component of Alzheimer's disease (AD) senile plaques in the brain is amyloid-beta peptide (Abeta), a proteolytic fragment of the amyloid precursor protein (APP). Platelets contain both APP and Abeta and much evidence suggests that these cells may represent a useful tool to study both amyloidogenic and nonamyloidogenic pathways of APP processing. It has been demonstrated that platelets activated by physiological agonists, such as thrombin and collagen, specifically secrete Abeta ending at residue 40. To verify whether APP beta-processing could be observed also in an in vitro system of highly concentrated platelets, we measured the Abeta released in the incubation media of 5 x 10(9) platelets/mL by enzyme-linked immunosorbent assay (ELISA). The activation status of platelets was investigated by ultrastructural analysis. We found that Abeta(40) levels were significantly higher in incubation media of 5 x 10(9)/mL platelets in comparison with 10(8)/mL platelets (normalized values), while Abeta(42) levels were not affected by cell density. The ultrastructural analysis showed platelets at different phases of activation: some platelets were at earlier stage, characterized by granule swelling and dilution, others had granules concentrated in a compact mass in the cell centers within constricted rings of circumferential microtubules (later stage). Normally concentrated cells had the characteristic morphology of resting platelets. Our data suggest that high-density platelets undergo activation likely by increased frequency of platelet-platelet collisions. This, in turn, determines the activation of APP beta-processing with consequent release of Abeta(40). Investigating the biochemical pathways triggering Abeta secretion in platelets might provide important information for developing tools to modulate this phenomenon in AD brains.
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There is increasing evidence that the cerebrovasculture may be involved in the pathology of Alzheimer's disease. Here, we report that potassium channel openers (KCOs) inhibit dose and time dependent necrosis induced by beta-amyloid (Abeta) in cultured vascular endothelial cells. Cell proliferation rate was assayed by a colorimetric method. Abeta cytotoxicity and inhibition by the K(ATP) channel opener diazoxide and the K(Ca) channel opener NS1619 was correlated with changes in nitric oxide (NO) production. The protective effects were partly blocked by potassium channel blockers. Toxicity of Abeta and KCO protection was verified by histological examination of endothelial cells with scanning electron microscopy. eNOS levels in endothelial cells were not changed by any of the treatments. The results suggest that disruption of K(+) channels function may be a critical step in Abeta-induced cytotoxicity in endothelial cells by alteration of NO release.
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K(+) channel openers can activate K channels and have been shown to protect cultured neurons against excitotoxicity. Our study showed that diazoxide, a K(+) channel opener, could counteract the effects of A beta(1-42) and protect cells from A beta(1-42)-induced the increasing of mitochondrial membrane potential and the associated increase in intracellular reactive oxygen species levels; an inducible nitric oxide synthase inhibitor, N omega-nitro-L-arginine could protect cells from A beta(1-42)-induced the increasing of both mitochondrial membrane potential and intracellular reactive oxygen species levels. A 24 h exposure to A beta(1-42) did not result in apoptosis, suggesting that the increase in both mitochondrial membrane potential and reactive oxygen species levels preceded cell apoptosis or death.
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Several lines of evidence indicate that Alzheimer's disease (AD) has systemic expression. Systemic changes are manifested as alterations in a number of molecular and cellular processes. Although, these alterations appear to have little or no consequence in peripheral systems, their parallel expression in the central nervous system (CNS) could account for the principal clinical manifestations of the disease. Recent research seems to indicate that alterations in ion channels, calcium homeostasis, and protein kinase C (PKC) can be linked and thereby constitute a model of pathophysiological relevance. Considering the difficulties of studying dynamic pathophysiological processes in the disease-ridden postmortem AD brain, peripheral tissues such as fibroblasts provide a suitable model to study molecular and cellular aspects of the disease.
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Alzheimer's disease is one of the most challenging brain disorders and has profound medical and social consequences. It affects approximately 15 million persons worldwide, and many more family members and care givers are touched by the disease. The initiating molecular event(s) is not known, and its pathophysiology is highly complex. However, free radical injury appears to be a fundamental process contributing to the neuronal death seen in the disorder, and this hypothesis is supported by many (although not all) studies using surrogate markers of oxidative damage. In vitro and animal studies suggest that various compounds with antioxidant ability can attenuate the oxidative stress induced by beta-amyloid. Recently, clinical trials have demonstrated potential benefits from treatment with the antioxidants, vitamin E, selegiline, extract of Gingko biloba, and idebenone. Further studies are warranted to confirm these findings and explore the optimum timing and antioxidant combination of such treatments in this therapeutically frustrating disease.
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In this paper, we describe an assay using radioactive rubidium (86Rb) efflux to screen functional human ether-a go-go-related gene (HERG) K+ channels in a high-throughput screening (HTS) format. This assay offers an alternative way to examine junctional interactions between chemical compounds and HERG K+ channels. Follow-up experiments and discussions were carried out to address a variety of factors that affect potency evaluation within the Rb efflux assay. Factors that can affect the assay results, such as assay time, efflux rate, and compound blocking kinetics, are discussed in detail. Our results provide some explanations for the variances of the assay results and offer some guidelines for using the Rb efflux assay to evaluate compound interactions with HERG K+ channels in the pharmaceutical industry.
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Low serum potassium increases risk of hypertension and stroke, and cardiovascular factors increase the risk of Alzheimer disease (AD). We examined the association between serum potassium and the biologic marker cerebrospinal fluid amyloid-beta (Abeta42), which is decreased in Alzheimer disease patients. Psychiatric examinations, laboratory and other tests were conducted on a population-based sample of 1080 women aged 46 to 60 in 1968, with follow-ups in 1974, 1980, and 1992. In 1992, cerebrospinal fluid Abeta42 levels were obtained from 81 women. Increasing serum potassium in 1968 was associated with increasing cerebrospinal fluid Abeta42 (beta = 153.9, P = 0.041) in 1992 using age-adjusted linear regression. Compared with the lowest tertile of potassium, the middle (beta = 95.3, P = 0.138) and highest tertiles (beta = 193.5, P = 0.004) had incrementally increased cerebrospinal fluid Abeta42 levels. Associations remained after controlling for blood pressure and other factors, and were similar among the 17 women in 1974 with available serum potassium. Potassium in 1980 and 1992 was not associated with cerebrospinal fluid Abeta42. Findings suggest low serum potassium in mid life, but not late life, is associated with low cerebrospinal fluid Abeta42 levels in late life. It is possible potassium co-varies with another variable that is associated with cerebrospinal fluid Abeta42. Nonetheless, serum potassium is a modifiable risk factor and further examination of the potassium-dementia relationship is warranted.
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We evaluated the antiplatelet effects of two classes of ATP-sensitive potassium channel openers (K(ATP) openers) on washed human platelets, and the study's emphasis was on the role of mitochondrial K(ATP) in platelet aggregation. Collagen-induced platelet aggregation was inhibited in a dose dependent manner by lemakalim and SKP-450, which are potent cardio-nonselective K(ATP) openers, and also by cardioselective BMS-180448 and BMS-191095 (IC50: 1,130, >1,500, 305.3 and 63.9 microM, respectively), but a significantly greater potency was noted for the cardioselective K(ATP) openers. The latter two K(ATP) openers also inhibited platelet aggregation induced by thrombin, another important blood-borne platelet activator, with similar rank order of potency (IC50: 498.0 and 104.8 microM for BMS-180448 and BMS-191095, respectively). The inhibitory effects of BMS-191095 on collagen-induced platelet aggregation were significantly blocked by a 30-min pretreatment of platelets with glyburide (1 microM) or sodium 5-hydroxydecanoate (5-HD, 100 microM), a nonselective and selective mitochondrial K(ATP) antagonist, respectively, at similar magnitudes; this indicates the role of mitochondrial K(ATP) in the antiplatelet activity of BMS-191095. However, glyburide and 5-HD had no effect when they were added to the platelet cuvette immediately prior to the addition of BMS-191095. These findings indicate that cardioselective mitochondrial K(ATP) openers like BMS-191095 are able to exert cardioprotective effects in cardiac ischemia/reperfusion injury via dual mechanisms directed at the inhibition of platelet aggregation and the protection of cardiomyocytes, and both these mechanisms are mediated by mitochondrial K(ATP).
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Numerous observations indicate that, while the predominant clinical expression arises from brain pathology, Alzheimer s disease (AD) has systemic expression at the cellular and molecular levels. Although these alterations seem to be inconsequential outside the central nervous system, their parallel expression in the brain could be considered a plausible pathophysiological model and explain part of the clinical manifestations; in particular those related to memory loss. Recent research has provided experimental evidence of a direct or indirect linkage between alteration in ion channels, PKC, calcium homeostasis and amyloid processing in peripheral tissues. Some evidence also indicates similar phenomena in the brain, attesting to the relevance of the changes in non CNS cells. Considering the difficulties of using post mortem material to study dynamic and/or early event in mostly end stage, disease ridden tissues, peripheral cells such as fibroblasts offer a model to study cellular aspects of AD pathophysiology.
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Potassium channel dysfunction has been implicated in a variety of genetic and acquired neurological disorders that are collectively referred to as the potassium channelopathies. These include acquired neuromyotonia, episodic ataxia type-1, hereditary deafness syndromes, benign familial neonatal convulsions and hypokalaemic periodic paralysis. Insight into potassium channel structure and function is crucial to understanding the pathophysiology of these conditions. This article describes potassium channel structure and function and then outlines what is known about the immunology and genetics of the neurological potassium channelopathies.
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In this paper we analyzed the operation of K+ channels by measuring the extracellular calcium influx in response to tetraethylammonium (TEA) depolarization. We compared TEA-induced intracellular calcium changes in fibroblasts obtained from Alzheimer Disease (AD) patients, aged (AC) and young (YC) healthy subjects. Subsequently we analyzed the effects of incubation of these fibroblasts with beta-amyloid on calcium responses. Our results indicated that depolarization with TEA did not abolish calcium responses in any of the tested cell groups. TEA-elicited calcium changes in AD cells were significantly greater than those observed in AC or YC cells. Responses were delayed in AD fibroblasts with respect to AC or YC cells. Incubation with beta-amyloid did not alter the other effects: response amplitude in AD cells was still greater than in AC or YC cells; AD responses were still delayed with respect to AC and YC responses. These results support the earlier findings that calcium responses were altered in AD cells but suggest their delay rather than inhibition.
Article
We have investigated using single channel patch-clamp methods potassium channel prevalence in hippocampal neurones from two animal models of AD. Experiments have been carried out on transgenic mice (Tg2576) carrying the Swedish mutation (K670N/M671L) and rats receiving ventricular infusions of okadaic acid. In cell-attached patches from hippocampal neurones from the Tg2576 and control littermate mice there were three principal unitary conductance - 22 pS, 111 pS and 178 pS. The two channels of intermediate and large conductance were voltage-dependent, highly active in cell-attached patches, activity decreasing markedly on hyperpolarisation. The large conductance channel was sensitive to TEA, iberiotoxin, was activated in excised inside-out patches by Ca 2+(i) and is the type I maxi-K+ channel. Significantly, there was a reduction in the prevalence of a TEA-sensitive 113 pS channel in neurones from TG2576 mice with a corresponding increase in prevalence of the maxi-K+ channel. There was no difference in the characteristics of maxi-K+ between patches in neurones from the transgenic and littermate controls. In the rat model single channel analysis was performed on hippocampal neurons from three groups of animals i.e. non-operated, and these receiving an infusion of vehicle or vehicle with okadaic acid. Three principal unitary conductances of around 18 pS, 118 pS and 185 pS were also observed in cell-attached recordings from these three groups. The intermediate and high conductance channels were blocked by TEA or 4-AP or 140 mM RbCl. There were no statistically significant differences in the channel prevalence or channel density between the control and test groups.
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Although lithium possesses neuroprotective functions, the molecular mechanism underlying its actions has not been fully elucidated. In the present paper, the effects of lithium chloride on voltage-dependent potassium currents in the CA1 pyramidal neurons acutely isolated from rat hippocampus were studied using the whole-cell patch-clamp technique. Depolarizing test pulses activated two components of outward potassium currents: a rapidly activating and inactivating component, I A and a delayed component, I K. Results showed that lithium chloride increased the amplitude of I A in a concentration-dependent manner. Half enhancement concentration (EC 50) was 22.80±5.45 μmol·L−1. Lithium chloride of 25 μmol·L−1 shifted the steady-state activation curve and inactivation curve of I A to more negative potentials, but mainly affected the activation kinetics. The amplitude and the activation processes of I K were not affected by lithium chloride. The effects of lithium chloride on potassium channel appear to possess neuroprotective properties by Ca2+-lowing effects modulate neuronal excitability by activating I A in rat hippocampal neurons.
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A liquid-liquid extraction separation procedure has been developed for the separation of trace amounts of neutron activated rubidium and strontium from their mixture in 0.1M perchloric acid medium using 0.04M 18-crown-6 in nitrobenzene as the extractant. Separation factor for Rb/Sr was found approximately 3.103. Gamma-radiation stability of the extractant (up to 1.5.104 Gy) as well as that of the metal-extractant complex were examined. Complete stripping of rubidium has been achieved by 8M HCl with 3–4 fold volume increase of the aqueous phase.
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Platelets participate in haemostasis and in thrombus formation in health and disease. Moreover, they contribute to inflammation and cooperate with immune cells in a magnitude of inflammatory/immune responses. Although the inflammatory response has been recognised to be critical in neuronal diseases such as Alzheimer´s disease or multiple sclerosis and its mouse counterpart, experimental autoimmune encephalomyelitis, the participation of platelets in these diseases is poorly investigated so far. Emerging studies, however, point to an interesting crosstalk between platelets and neuroinflammation. For instance, when the integrity of the blood brain barrier is compromised, platelets may be relevant for endothelial inflammation, as well as recruitment and activation of inflammatory cells, thereby potentially contributing to central nervous tissue pathogenesis. This review summarises recent insights in the role of platelets to neurovascular inflammation and addresses potential underlying mechanisms, by which platelets may affect the pathophysiology of neurovascular diseases.
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Seventeen otherwise healthy, non-smoking patients with probable Alzheimer's disease (AD) and 11 healthy, non-smoking, age-matched controls participated in a study of vascular reactivity. Three substances were iontophoresed into the skin: sodium nitroprusside (SNP) delivered at the cathode, and acetylcholine chloride and isoprenaline sulphate delivered at the anode. The resultant vasodilation was mapped through a laser Doppler perfusion imager. The patients harbouring the Apolipoprotein E ε4 allele (ApoE4) (n=9) showed significantly greater vasodilation compared both with patients without the ApoE4 and with controls (one control possessed the ApoE4) when SNP was delivered at the cathode. An additional study of ten of the AD patients and ten controls suggested that the enhanced reactivity in the patients with the ApoE4 depended on a greater sensitivity to the iontophoretic current itself at the cathode rather than to SNP. The quotient between total cholesterol and high-density lipoprotein-cholesterol was higher in the patients with the ApoE4 compared both with patients without the ApoE4 and with controls. A dysfunction of voltage-dependent ion channels and/or other membrane abnormalities in AD patients possessing the ApoE4 is suggested.
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Introduction: Alzheimer’s disease is a progressive neurodegenerative disorder, characterized by impairment of memory and changes in behavior and personality. Recent evidence suggests that mitochondrial channels play important roles in memory disorders. Accordingly, the biophysical properties of a single potassium channel were investigated in the brain mitochondrial inner membrane of rat with Alzheimer’s disease. Methods: In the male Wistar rats (220- 250 g), Alzheimer’s disease was induced by intracerebroventricular injection of amyloid beta 1-42 (4μg/μL). After two weeks, the brain mitochondrial inner membranes were extracted. Vesicles were incorporated into lipid bilayer membranes, and single potassium channel properties were investigated. Also, purity of the cell fraction was tested by Western blotting. Protein samples were probed with specific antibodies. Results:based on previous data, mitochondrial inner membrane has a potassium channel with a main conductance 93 pS which was 4-APsensitive and voltage-insensitive at -50 to +40mV. In the present study, it was demonstrated that the channel conductance was increased to 114 pS in Alzheimer's Disease . In addition,the current - voltage relationship showed an inward rectification. western blotting and antibodies directed against various cellular proteins revealed that the extracted material contains only mitochondria. Conclusion: Our data showed that the biophysical properties (gating, conductance and activities) of potassium channel were significantly altered in Alzheimer’s disease. Based on these findings, we propose that the brain mitochondrial potassium channels are involved in Alzheimer’s disease, and it can be considered as a target for therapeutic plans.
Article
Background: There was only one foreign laboratory observed the result of a high positive rate change of erythrocyte electrophoresis in Alzheimer disease. Objective: To research the relationship between time of erythrocyte electrophoresis and Alzheimer disease as diagnostic basis for clinical assay of Alzheimer disease. Design: Case controlled clinical single blind trial. Setting and participants: A total of 11 patients diagnosed as Alzheimer disease, above 60 years old among communities in Beijing, 13 patients were vascular dementia group, and 53 patients were control group without dementia and cerebral infarction. Interventions: The change of time of erythrocyte electrophoresis in those two groups was observed, and statistical analysis was used. Main outcome measures: Time of erythrocyte electrophoresis and positive rate of diagnosis. Results: Time of erythrocyte electrophoresis in Alzheimer disease patients is less than that of in aged group, with 90% single lower limit value i. e. 12.74 s, as the positive verge, the positive rates in Alzheimer disease group, vascular dementia group and aged control group were 81.82% (P = 0.000 001 23 < 0.001), 76.92% (P = 0.000 01024 < 0.001), and 7.55%, respectively. Conclusion: Shortening of time of erythrocyte electrophoresis is related to Alzheimer disease, and it is very specific for the diagnosis of Alzheimer disease.
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By measuring intracellular Ca2+ release and K+ channel defects in fibre-blasts, we have refined experimental parameters in order to obtain a more definitive diagnostic assay for AD. Genetic studies have previously established that individuals who have certain mutations in the Alzheimer's disease (AD) gene known as presenilin-1 will eventually become symptomatic for AD. Here, we investigated whether pathophysiological defects are manifested in fibroblasts of these gene carriers for AD prior to the occurrence of symptoms. Using fluorescent imaging techniques, we studied fibroblasts from 21 patients with AD, 15 age-matched controls and 18 individuals from families carrying presenilin-1 mutations. A 'ratio index1, derived from IPS-mediated Ca2+ release in relation to K+ channel closure-induced Ca2+ influx, was obtained for each cell line. The ratio index was able to completely distinguish AD from age-matched controls. The index values for pre-symptomatic presenilin-1 (mutation carriers) was within the range of the AD patients. Conversely, kin of the same AD families who do not carry the mutation had index values within the range of the age matched-control group. These results demonstrate that the ratio index diagnostic for AD can predict the subsequent occurrence of AD in asymptomatic individuals who carry a mutation in the presenilin gene.
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Potassium ion (K+) plays a central role in several fundamental physiological processes. Detection of the K+ concentration is an essential diagnostic tool for various medical diseases. However, most commercial detection methods are complex and expensive, which are not easily implemented in community hospitals or at home, in this study, we present a simple fluorescent K+ detection system based on the formation of G-quadruplex between K+ and dual-labelled thrombin aptamer oligonucleotide derivative (5′-FAM-TTTTTTAGGTTGGTGTGGTTGG-TAMRA-3′). Furthermore, based on this method, highly sensitive and selective detection of K+ in actual serum was realized by using EDTA as chelating agent to avoid the interference of Ca2+ and Mg2+ at physiological concentrations. Thus, this study paves the road toward the design and manufacture of portable potassium ions sensors based on fluorescence.
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Rapid and sensitive detection of thrombin is imperative for the early diagnosis, prevention, and treatment of thrombin‐related diseases. Here, an ultrasensitive and rapid thrombin biosensor is developed based on rationally designed trifunctional protein HTs, comprising three functional units, including a far‐red fluorescent protein smURFP, hydrophobin HGFI, and a thrombin cleavage site (TCS). smURFP is used as a detection signal to eliminate any interference from the autofluorescence of sample matrix to increase detection sensitivity. HGFI serve as an adhesive unit to allow rapid immobilization of HTs on a multiwall plate. The TCS linking HGFI and smURFP function as a sensing element to recognize and detect thrombin. HTs immobilization is symmetrically optimized and characterized. Thrombin assay reveals the specific recognition of active thrombin in samples and the hydrolysis of the immobilized HTs, resulting in a decrease in the fluorescence intensity of the sample in a thrombin concentration‐dependent manner. The limit of detection (LOD) is as low as 0.2 am in the serum. To the authors’ knowledge, this is the lowest LOD ever reported for any thrombin biosensor. This study sheds light on the engineering of multifunctional proteins for biosensing.
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Alzheimer's disease (AD) is the most common neurodegenerative disease characterized clinically by progressive memory loss and decline in cognitive abilities and characterized pathologically by the presence of two types of abnormal deposits, i.e., senile plaques (SP) and neurofibrillary tangles (NFT), and by extensive synapse and neuronal loss. SP are composed of fibrillar amyloid beta-peptide (Abeta) surrounded by dystrophic neurites. Recent studies suggest two prospective mechanisms for Abeta-associated membrane dysfunction and subsequent neurotoxicity. One suggests that Abeta oligomers can form heterogeneous ion-channels in the cell membrane leading to cellular degeneration, while the second suggests insertion of Abeta oligomers in membrane lipid bilayers could induce the dysfunction of ion-channels or pumps by binding to or inducing oxidative modification of membrane proteins. In this review, we discuss the effects of Abeta on membrane proteins that are involved in cholinergic and glutamatergic pathways, and some ion-channels.
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A combination of medial temporal lobe atrophy, shown by computed tomography, and reduced blood flow in the parietotemporal cortex, shown by single photon emission tomography, was found in 86% (44/51) of patients with a clinical diagnosis of senile dementia of the Alzheimer type (SDAT). The same combination of changes was found in four out of 10 patients with other clinical types of dementia and in two out of 18 with no evidence of cognitive deficit. Of the 12 patients who died, 10 fulfilled histopathological criteria for Alzheimer's disease, nine of them having a clinical diagnosis of SDAT, and one a clinical diagnosis of multi-infarct dementia. All 10 patients with histopathologically diagnosed Alzheimer's disease had shown a combination of hippocampal atrophy and reduced parietotemporal blood flow in life. In 10 patients (nine with SDAT) out of 12 in whom the hippocampal atrophy was more noticeable on one side of the brain than on the other the parietotemporal perfusion deficit was also asymmetrical, being greater on the side showing more hippocampal atrophy. These results suggest that the combination of atrophy of the hippocampal formation and reduced blood flow in the parietotemporal region is a feature of dementia of the Alzheimer type and that the functional change in the parietotemporal region might be related to the loss of the projection neurons in the parahippocampal gyrus that innervate this region of the neocortex.
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Since memory loss is characteristic of Alzheimer disease (AD), and since K+ channels change during acquisition of memory in both molluscs and mammals, we investigated K+ channel function as a possible site of AD pathology and, therefore, as a possible diagnostic index as well. A 113-pS tetraethylammonium (TEA)-sensitive K+ channel was consistently absent from AD fibroblasts, while it was often present in young and aged control fibroblasts. A second (166-pS) K+ channel was present in all three groups. Elevated external potassium raised intracellular Ca2+ in all cases. TEA depolarized and caused intracellular Ca2+ elevation in young and aged control fibroblasts but not AD fibroblasts. The invariable absence of a 113-pS TEA-sensitive K+ channel and TEA-induced Ca2+ signal indicate K+ channel dysfunction in AD fibroblasts. These results suggest the possibility of a laboratory method that would diagnostically distinguish AD patients, with or without a family history of AD, from normal age-matched controls and also from patients with non-AD neurological and psychiatric disorders.
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1. Na⁺/K⁺/2Cl⁻ co-transport mediates a bidirectional symport of Na⁺, K⁺ and Cl⁻. The important properties of the co-transport system are its requirement for Na⁺, K⁺ and Cl⁻ and its inhibition by loop diuretics such as bumetanide. This co-transporter has been described in a number of animal and human tissues. However, its presence in human platelets, although inferred, has not been demonstrated directly. 2. We have studied the efflux of ⁸⁶Rb⁺ (a marker for K⁺) from Rb⁺-loaded platelets, and have defined their response to stimulation by high concentrations of external K⁺. 3. KCl (30–120 mmol/l) stimulated a concentration-dependent increase in ⁸⁶Rb⁺ efflux from the platelets. This efflux was completely inhibited by bumetanide (10 μmol/l) but was insensitive to ouabain and R(+)-[(dihydroindenyl)oxy]alkanoic acid. It also required Cl⁻ in the external medium, but did not depend on the presence of extracellular Na⁺. 4. These observations suggest that ⁸⁶Rb⁺ efflux from platelets stimulated by external K⁺ occurs via Na⁺/K⁺/2Cl⁻ co-transport acting in a K⁺/K⁺ (K⁺/Rb⁺) exchange mode. 5. Non-stimulated efflux of ⁸⁶Rb⁺ from the platelets (i.e. in the presence of 5 mmol/l K⁺) had the characteristics of Na⁺/K⁺/2Cl⁻ co-transport acting in normal mode.
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In a prospective study of more than 200 cases of dementia and 119 controls, annual technetium-99mnon-ADAlzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) and the DiagnosticandStatisticalManualofMentalDisorders (3rd ed., rev.; DSM-III-R) criteria, and all histopathological diagnoses according to the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) criteria. Early data from this cohort have suggested that the combination of both MTL atrophy seen on CT with parietotemporal hypoperfusion on SPECT may predict the pathology of AD. The diagnostic sensitivity, specificity, accuracy, and positive and negative predictive values of the NINCDS-ADRDA and DSM-III-R criteria could be assessed in this cohort against the gold standard of histopathology. The diagnostic potential of CT evidence of MTL atrophy alone, SPECT evidence of parietotemporal hypoperfusion alone, and the combination of both of these scan changes in the same individual could then be compared against the diagnostic accuracy of clinical operational criteria in the pathologically confirmed cases. Furthermore, all of these modalities could be compared with the diagnostic accuracy of apolipoprotein E4 (Apo E4) genotyping to predict AD in the histopathologically confirmed cohort. In this population, NINCDS was 100% specific, 49% sensitive, and 66% accurate; was only 61% specific, but 93% sensitive and 77% accurate; and the combination of both and possible-ADnon-AD” dementias later in the course of the disease suggests the concept of medial temporal lobe dementia. This could explain some of the overlap of clinical profiles in the dementias, particularly as the dementia progresses, making clinical differential diagnosis difficult. In this context, the use of SPECT can significantly enhance specificity.
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The medial temporal lobe of the brain is important for normal cognitive function, notably for memory, and is the region with the most extensive pathological change in Alzheimer's disease (AD). We wanted to find out if atrophy of the medial temporal lobe could be detected in life in patients in whom a diagnosis of AD was subsequently established histopathologically. The minimum width of the medial temporal lobe, measured by temporal-lobe-oriented computed tomography (CT) about one year before death, in 44 patients with a histopathological diagnosis of AD (cases) was nearly half (0.56 of the median) that in 75 controls of the same age with no clinical evidence of dementia (95% confidence interval 0.51-0.61). There was little overlap between the distributions of measurements in cases and controls. A cut-off (< 0.79 MoM) selected to yield a 5% false-positive rate gave an expected detection rate of 92%. A cut-off selected to yield a false-positive rate of 1% (< 0.70 MoM) yielded a 79% detection rate. 20 of the 44 patients with histopathologically diagnosed AD had been scanned more than once before death, and the test (cut-off < 0.79 MoM) was positive in all 20 more than a year before and in 9/10 more than 2 years before death. In 10 subjects with dementia but with histopathology excluding AD, the mean minimum width of the medial temporal lobe was significantly greater than that in the cases with AD, but was not significantly different from that in controls. Medial temporal lobe CT is a non-invasive, rapid, simple and effective test for AD which could have immediate application firstly in improving the accuracy of prevalence and incidence studies and, secondly, for the identification of groups of high-risk patients in the evaluation of novel treatments for AD. In the future, it could be applied as a screening test.
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[125I]Apamin binding sites were examined using quantitative autoradiography in the hippocampus of 9 patients with Alzheimer's disease and 8 age-matched controls. Within the hippocampal formation from control subjects, [125I]apamin binding sites were highly concentrated in the subiculum and CA1. In Alzheimer's disease there was a marked and discrete loss of [125I]apamin binding sites in the subiculum (control = 1.10 +/- 0.10 pmol/g; Alzheimer = 0.71 +/- 0.09 pmol/g) and CA1 (control = 1.41 +/- 0.09 pmol/g; Alzheimer = 0.85 +/- 0.11 pmol/g; values are mean +/- S.E.M.). This reduction of [125I]apamin binding sites in the subiculum correlated with cell density but not neuritic plaque density. These results indicate that an anatomically discrete loss of Ca(2+)-dependent K+ channels within the hippocampal formation occurs in Alzheimer's disease.
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In order to study the ionic efflux or granule release from human platelets following pulse exposure to various stimuli, a method for continuous perfusion of platelets was developed. The method was applied to compare the effects of membrane depolarization and thrombin stimulation on the release of 86Rb and [3H]5-HT. Washed and preloaded human platelets were placed on a membrane filter in a temperature controlled polypropylene chamber, and subsequently perfused with buffer. After an initial washout period the efflux of 86Rb or [3H]5-HT reached steady, low levels. K+ induced concentration dependent increases in 86Rb efflux, corresponding to a depolarization of the membrane potential, whereas the efflux of [3H]5-HT was unaltered. Thrombin induced concentration dependent increases in the efflux of both 86Rb and [3H]5-HT. Pretreatment with K+ 12 or 30 mM did not alter the [3H]5-HT efflux induced by thrombin 0.1 U ml-1. Scanning electron micrographs of platelets on the filter showed that the unstimulated platelets had regular shape, whereas after addition of thrombin there was formation of pseudopods and minor aggregates. The effect of potassium-induced membrane depolarization on platelet aggregation was also studied. High concentration of K+ did not induce aggregation or shape change during 2 or 10 minutes of incubation. K+ had little or no effect on aggregation induced by ADP 2 microM or thrombin 0.4 U ml-1. The results from release experiments and aggregation tests argue against an immediate coupling between membrane potential and platelet reactivity.
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Protease nexin-II (PN-II) [amyloid beta-protein precursor (APP)] and the amyloid beta-protein are major constituents of neuritic plaques and cerebrovascular deposits in individuals with Alzheimer's disease and Down syndrome. Both the brain and the circulation have been implicated as sources of these molecules, although they have not been detected in blood. Human platelets have now been found to contain relatively large amounts of PN-II/APP. Platelet PN-II/APP was localized in platelet alpha-granules and was secreted upon platelet activation. Because PN-II/APP is a potent protease inhibitor and possesses growth factor activity, these results implicate PN-II/APP in wound repair. In certain disease states, alterations in platelet release and processing and clearance of PN-II/APP and its derived fragments could lead to pathological accumulation of these proteins.
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Previous reports have suggested that the physical properties of cell membranes and calcium homeostasis in both the central and peripheral nervous system are changed in Alzheimer's disease (AD). This study has examined the biophysical properties of erythrocyte and platelet membranes by measuring the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and possible related changes in lipid peroxidation. In addition, we have studied calcium homeostasis by measuring thrombin-stimulated changes in intraplatelet free calcium and Ca2(+)-ATPase activity in AD and healthy age and sex-matched controls. Our results show that there was no significant difference in the fluorescence anisotropy of DPH in erythrocyte membranes isolated from the three groups. There was also no significant difference in lipid peroxidation levels in erythrocytes and plasma of AD patients compared to controls. However, there was a significant reduction in the fluorescence anisotropy of DPH in platelet membranes from AD patients, compared with healthy controls. Recent evident suggests that the increase in platelet membrane fluidity results from alterations in internal membranes. We measured the specific activities of enzyme markers associated with intracellular and plasma membranes in platelets from AD patients and healthy controls. There was a significant reduction in the specific activity of antimycin A-insensitive NADH-cytochrome-c reductase (a specific marker for smooth endoplasmic reticulum (SER)), in AD patients compared to controls, but no change in the specific activity of bis(p-nitrophenyl)phosphate phosphodiesterase (a specific marker for plasma membrane). We have also shown that SER mediated [Ca2+] homeostasis is possibly impaired in AD platelets, i.e., the percentage of thrombin-stimulated increase in intraplatelet [Ca2+] above basal levels was significantly higher in AD compared to matched controls and there were significant reductions in the specific activities of Ca2+/Mg2(+)-ATPase and Ca2(+)-ATPase (but not Mg2(+)-ATPase) in AD platelets. Finally electron microscopic analysis of platelets showed that there was a significant increase in the incidence of abnormal membranes in AD patients compared to controls. The ultrastructural abnormalities seem to consist of proliferation of a system of trabeculated cisternae bounded by SER. These results suggest that both SER structure and function might be defected in AD platelets, which could explain the fluidity changes observed here.
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A new interview schedule for the diagnosis and measurement of dementia in the elderly is described. The schedule named the Cambridge Mental Disorders of the Elderly Examination (CAMDEX), consists of three main sections: A structured clinical interview with the patient to obtain systematic information about the present state, past history and family history; a range of objective cognitive tests which constitute a mini-neuropsychological battery; a structured interview with a relative or other informant to obtain independent information about the respondent's present state, past history and family history. The CAMDEX is acceptable to patients, has a high inter-rater reliability and the cognitive section has been shown to have high sensitivity and specificity.
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Clinical criteria for the diagnosis of Alzheimer's disease include insidious onset and progressive impairment of memory and other cognitive functions. There are no motor, sensory, or coordination deficits early in the disease. The diagnosis cannot be determined by laboratory tests. These tests are important primarily in identifying other possible causes of dementia that must be excluded before the diagnosis of Alzheimer's disease may be made with confidence. Neuropsychological tests provide confirmatory evidence of the diagnosis of dementia and help to assess the course and response to therapy. The criteria proposed are intended to serve as a guide for the diagnosis of probable, possible, and definite Alzheimer's disease; these criteria will be revised as more definitive information become available.
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The main component of Alzheimer's disease (AD) amyloid deposits is amyloid beta-peptide (A beta), a fragment of the larger amyloid precursor protein (APP). The cellular source of A beta is not known, but a circulatory origin has been postulated. We studied human blood from healthy individuals and found that platelets account for almost 90% of the total anti-A beta immunoreactivity detected in whole blood. Using reverse-phase HPLC, we identified a platelet peptide which corresponds to A beta by three criteria: (a) it shares a retention time with the synthetic A beta 1-40 peptide in two consecutive HPLC tests; (b) it interacts with two anti-A beta antibodies in separate ELISAs; and, (c) its partial N-terminal amino acid sequence closely matches that of A beta. The detection of this peptide in platelets indicates that, aside from the well-known non-amyloidogenic (secretory) pathway, the processing of APP in platelets from healthy individuals also involves an amyloidogenic pathway. These findings are consistent with the view that platelets are one of the major sources of A beta in the circulation.
Article
The most common cause of dementia in the developed world is Alzheimer's disease. Histopathology is required to confirm diagnosis, but most evaluations of the accuracy of clinical criteria and neuroimaging in the diagnosis of dementia of the Alzheimer type are without such confirmation. The average specificity of clinical criteria alone is about 75%. This paper discusses the contribution of simple structural (x-ray computed tomography [CT]) and functional (Tc-99m-HMPAO single photon emission computed tomography [SPECT]) imaging to the diagnosis of Alzheimer's disease in 71 histopathologically confirmed cases (47 with Alzheimer's disease, 16 with other dementias, 8 controls) and 84 living controls. Medial temporal lobe atrophy assessed by temporal lobe-oriented CT gave 94% sensitivity and 93% specificity, while parietotemporal hypoperfusion on SPECT revealed 96% sensitivity and 89% specificity. The combination of both changes yielded a sensitivity of 90% and a specificity of 97%. These investigations clearly enhance diagnostic accuracy, can be readily applied in the clinical situation, and could be used in epidemiologic studies of Alzheimer's disease.
Article
Although beta-amyloid is the main constituent of neurite plaques and may play a role in the pathophysiology of Alzheimer's disease, mechanisms by which soluble beta-amyloid might produce early symptoms such as memory loss before diffuse plaque deposition have not been implicated. Treatment of fibroblasts with beta-amyloid (10 nM) induced the same potassium channel dysfunction previously shown to occur specifically in fibroblasts from patients with Alzheimer's disease--namely, the absence of a 113-picosiemen potassium channel. A tetraethylammonium-induced increase of intracellular concentrations of calcium, [Ca2+]i, a response that depends on functional 113-picosiemen potassium channels, was also eliminated or markedly reduced by 10 nM beta-amyloid. Increased [Ca2+]i induced by high concentrations of extracellular potassium and 166-picosiemen potassium channels were unaffected by 10 nM beta-amyloid. In Alzheimer's disease, then, beta-amyloid might alter potassium channels and thus impair neuronal function to produce symptoms such as memory loss by a means other than plaque formation.
Article
1The pharmacological characteristics of a putative Ca2+ activated K+ channel (IKCa channel) in rat glioma C6 cells were studied in the presence of the Ca2+ ionophore, ionomycin and various K+ channel blockers, 86Rb+ being used as a radioisotopic tracer for K+.2The resting 86Rb+ influx into C6 cells was 318±20 pmol s−1. The threshold for ionomycin activation of 86Rb+ influx was approx. 100 nM. At ionomycin concentrations above the activation threshold, the initial rate of 86Rb+ influx was proportional to ionophore concentration. Ionomycin-activated 86Rb+ flux was saturable (EC50 = 0.62±0.03 μm) and was not inhibited by ouabain.3Intracellular Ca2+ increased within 30 s from a basal level of 42±2 nM to 233±17 nM, after addition of 2 μm ionomycin. During this period, intracellular pH fell from 7.03±0.04 to 6.87±0.03 and the cell hyperpolarized from −34±10 mV to −76±2 mV.4Single channel conductance measurements on inside-out patches in physiological K+ solutions identified a 14±3 pS Ca2+ -activated K+ current between −25 mV and +50 mV. In symmetrical (100 mM) K+, the single channel conductance was 26 pS.5Externally applied quinine (IC50 = 0.12±0.34 mM) and tetraethylammonium chloride (IC50 = 10±1.9 mM) inhibited 86Rb+ influx into C6 cells in a concentration-dependent manner. Charybdotoxin (IC50 = 0.5±0.02 nM) and iberiotoxin (IC50 = 800±150 nM), as well as the crude venoms from the scorpions Leiurus quinquestriatus and Mesobuthus tamulus, also inhibited 86Rb+ influx. In contrast, apamin and toxin I had no inhibitory effects on 86Rb+ flux. A screen of fractions from cation exchange h.p.l.c. of Mesob. tamulus venom revealed the presence of at least four charybdotoxin-like peptides. One of these was iberiotoxin; the other three are novel toxins.6The ionomycin-activated 86Rb+ influx into rat C6 glioma cells has proved to be a valuable pharmacological assay for the screening of toxins and crude venoms which modify intermediate conductance, Ca2+ activated K+ channel activity.
Article
Alzheimer's disease is a progressive dementia characterized in part by deposition of proteinaceous plaques in various areas of the brain. The main plaque protein component is beta-amyloid, a metabolic product of the beta-amyloid precursor protein. Substantial evidence has implicated beta-amyloid (and other amyloidogenic fragments of the precursor protein) with the neurodegeneration observed in Alzheimer's disease. Recently, beta-amyloid precursor protein and its amyloidogenic metabolic fragments have been shown to alter cellular ionic activity, either through interaction with existing channels or by de novo channel formation. Such alteration in ionic homeostasis has also been linked with cellular toxicity and might provide a molecular mechanism underlying the neurodegeneration seen in Alzheimer's disease.
Article
1. Previous electrophysiological studies have suggested the presence of KCa and Kv channels in human platelets. However, the pharmacology of these channels has not been defined. 2. We have studied potassium channels in human platelets by measuring the efflux of 86Rb+ (a marker for K+) from 86Rb+-loaded cells, and have defined their responses to stimulation by the platelet agonist thrombin and the calcium ionophore ionomycin. 3. Thrombin (0.1–0.6 i.u./ml) stimulated an increase in 86Rb+ efflux from the platelets in a concentration-dependent manner. This efflux was significantly inhibited by apamin (100 nmol/l), charybdotoxin (300 nmol/l) and α-dendrotoxin (100–200 nmol/l), blockers of SKCa channels, KCh channels and Kv channels respectively. Iberiotoxin (300 nmol/l), a specific inhibitor of BKCa channels, had no effect on the thrombin-stimulated 86Rb+ efflux. Although glibenclamide, an inhibitor of KATP channels, inhibited the thrombin-stimulated efflux, it did so only in a high concentration (20 μmol/l). 4. Ionomycin (1–5 μmol/l) stimulated an increase in 86Rb+ efflux from the platelets in a concentration-dependent manner. This efflux was significantly inhibited by apamin (100 nmol/l) and charybdotoxin (300 nmol/l). However, iberiotoxin (300 nmol/l) had no effect on the ionomycin-stimulated 86Rb+ efflux. 5. These findings suggest that 86Rb+ efflux from platelets stimulated by thrombin and ionomycin occurs via two types of KCa channel: SKCa and KCh channels. Thrombin also stimulated efflux via Kv channels.
Article
In a prospective study of more than 200 cases of dementia and 119 controls, annual technetium-99m-hexamethyl-propylene amineoxime (99mTc-HMPAO) single-photon emission computed tomography (SPECT) and annual medial temporal lobe (MTL) oriented X-ray computed tomography (CT) have been used to evaluate the diagnostic potential of functional and structural neuroimaging in the differential diagnosis of dementia. Some subjects have had up to 7 annual evaluations. So far, of 151 who have died, 143 (95%) have come to necropsy. Histology is known for 118, of whom 80 had Alzheimer's disease (AD), 24 had other "non-AD" dementias, and 14 controls with no cognitive deficit in life also had no significant central nervous system pathology. To compare the findings in the dementias with the profile of structural and functional imaging in the cognitively normal elderly, scan data from 105 living, elderly controls without cognitive deficit have also been included in the analysis. All clinical diagnoses were according to National Institute of Neurological and Communicable Disease and Stroke-Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) and the Diagnostic and Statistical Manual of Mental Disorders (3rd ed., rev.; DSM-III-R) criteria, and all histopathological diagnoses according to the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) criteria. Early data from this cohort have suggested that the combination of both MTL atrophy seen on CT with parietotemporal hypoperfusion on SPECT may predict the pathology of AD. The diagnostic sensitivity, specificity, accuracy, and positive and negative predictive values of the NINCDS-ADRDA and DSM-III-R criteria could be assessed in this cohort against the gold standard of histopathology. The diagnostic potential of CT evidence of MTL atrophy alone, SPECT evidence of parietotemporal hypoperfusion alone, and the combination of both of these scan changes in the same individual could then be compared against the diagnostic accuracy of clinical operational criteria in the pathologically confirmed cases. Furthermore, all of these modalities could be compared with the diagnostic accuracy of apolipoprotein E4 (Apo E4) genotyping to predict AD in the histopathologically confirmed cohort. In this population, NINCDS "probable-AD" was 100% specific, 49% sensitive, and 66% accurate; "possible-AD" was only 61% specific, but 93% sensitive and 77% accurate; and the combination of both "probable-AD" and "possible-AD" was 61% specific, 96% sensitive, and 85% accurate. DSM-III-R criteria were 51% sensitive, 97% specific, and 66% accurate. In the same cases and including the 105 living, elderly controls, the diagnostic accuracy of the Oxford Project to Investigate Memory and Aging (OPTIMA) scanning criteria showed CT alone to be 85% sensitive, 78% specific, and 80% accurate; SPECT alone had 89% sensitivity, 80% specificity, and 83% accuracy; and the combination of the two was 80% sensitive, 93% specific, and 88% accurate. The Apo E4 genotype was 74% sensitive but yielded 40% false positives in the histologically confirmed series. The diagnostic accuracy afforded by this method of CT and SPECT used alone is better than that of any established clinical criteria and reveals that the combination of MTL atrophy and parietotemporal hypoperfusion is common in AD, much less common in other dementias, and rare in normal controls. In the NINCDS-ADRDA criteria "possible-AD" cases, the combination of CT and SPECT findings alone were better in all diagnostic indices than the presence of Apo E4 alone in predicting AD. The frequent occurrence of MTL atrophy in AD and also in other "non-AD" dementias later in the course of the disease suggests the concept of medial temporal lobe dementia. This could explain some of the overlap of clinical profiles in the dementias, particularly as the dementia progresses, making clinical differential diagnosis difficult. In this context, the use of SPECT can significantly e
CAMDEX, the Cambridge examination for mental disorders of the elderly
  • M Roth
  • F A Huppert
  • E Tym
  • C Q Mountjoy
Roth M, Huppert FA, Tym E, Mountjoy CQ. CAMDEX, the Cambridge examination for mental disorders of the elderly. Cambridge: Cambridge University Press, 1988.