The Association of Blood-Based Inflammatory Factors IL-1β, TGF-β and CRP with Cognitive Function in Alzheimer’s Disease and Mild Cognitive Impairment

Objective Many patients suffer from dementia in its most common form, Alzheimer’s disease (AD). In this study, the levels of IL-1β, TGF-β and CRP, which are involved in the inflammatory response in Alzheimer’s disease and its mild cognitive impairment (MCI), were measured and analyzed. Methods Seventy nine subjects participated in this study (mean age: 75.56 years, female: 54.3%, AD: 26, MCI: 28, normal: 25). The overall cognitive function of the subjects and the severity of the disease stage were assessed using the Mini-Mental State Examination (MMSE-K), the Clinical Dementia Rating (CDR), the Global Deterioration Scale (GDS) and the Geriatric Depression Scale-Korean (GDS-K). Results It was observed that patients with AD had significantly higher levels of IL-1β and TGF-β than the patients with MCI and normal controls. In addition, the MCI group showed a statistically significantly higher TGF-β concentration than the normal group. Conclusion These results suggest that IL-1β and TGF-β may be useful biological markers for patients with Alzheimer’s disease.     

α2 and α1-adrenoceptors mediate opposing actions on parasympathetic neurons

We used intracellular recording methods to analyze the membrane responses to norepinephrine in cat vesical parasympathetic ganglia. In parasympathetic neurons, norepinephrine (NE) produces a membrane hyperpolarization, a membrane depolarization often accompanied by cell firing and a biphasic potential, a hyperpolarization followed by a depolarization. We found that the NE hyperpolarization is mediated through alpha 2-adrenoceptors while the NE depolarization is mediated through alpha 1-adrenoceptors. This situation is different than in sympathetic neurons where beta-adrenoceptors mediate a NE depolarization.     

Attenuation of β‐amyloid‐induced tauopathy via activation of CK2α/SIRT1: Targeting for cilostazol

β‐Amyloid (Aβ) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2‐coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac‐tau) and tau phosphorylation (P‐tau) by inhibiting activation of P300 and GSK3β. Aβ was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aβ accumulation was accompanied by increased Ac‐tau and P‐tau levels. Concomitantly, these cells showed increased P300 and GSK3β P‐Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3–30 μM) and resveratrol (20 μM). Moreover, decreased expression of SIRT1 and its activity by Aβ were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 μM, PKA inhibitor), TBCA (20 μM, inhibitor of CK2), and sirtinol (20 μM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP‐dependent protein kinase‐linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau‐related neurodegeneration in the AD brain. © 2013 Wiley Periodicals, Inc.     

Transglutaminase facilitates the formation of polymers of the β-amyloid peptide

One of the major pathological characteristics of Alzheimer's disease is the increased number of amyloid-containing senile plaques within the brain. The dense cores of these plaques are composed primarily of highly insoluble aggregates of a 39–43-residue peptide referred to as the β-amyloid peptide (βA). The mechanisms by which these insoluble extracellular deposits of βA are formed remain unknown. In this study, the cross-linking of βA by the calcium-dependent enzyme, transglutaminase was examined. Transglutaminases are a family of enzymes which are found in brain, and catalyse the cross-linking of specific proteins into insoluble polymers. Synthetic βA (1–40) was readily cross-linked by transglutaminase, forming multimers in a time-dependent fashion. Furthermore, a second peptide with a substitution similar to that in the Dutch-type hereditary amyloidosis mutation (Glu²² to Gln) was also found to be a substrate fro transglutaminase. Since transglutaminase covalently cross-links proteins through glutamine residues, it is suggested that transglutaminase contributes to amyloid deposition in Dutch-type hereditary amyloidosis, and possibl Alzheimer's disease.     

Restoring Soluble Amyloid Precursor Protein α Functions as a Potential Treatment for Alzheimer's Disease

Soluble amyloid precursor protein α (sAPPα), a secreted proteolytic fragment of nonamyloidogenic amyloid precursor protein (APP) processing, is known for numerous neuroprotective functions. These functions include but are not limited to proliferation, neuroprotection, synaptic plasticity, memory formation, neurogenesis, and neuritogenesis in cell culture and animal models. In addition, sAPPα influences amyloid‐β (Aβ) production by direct modulation of APP β‐secretase proteolysis as well as Aβ‐related or unrelated tau pathology, hallmark pathologies of Alzheimer's disease (AD). Thus, the restoration of sAPPα levels and functions in the brain by increasing nonamyloidogenic APP processing and/or manipulation of its signaling could reduce AD pathology and cognitive impairment. It is likely that identification and characterization of sAPPα receptors in the brain, downstream effectors, and signaling pathways will pave the way for an attractive therapeutic target for AD prevention or intervention. © 2016 Wiley Periodicals, Inc.     

Scavenging of Alzheimer's amyloid β-protein by microglia in culture

Deposits of amyloid β-protein (Aβ) form the cores of the pathological plaques which characterize Alzheimer's disease. The mechanism of formation of the deposits is unknown; one possibility is failure of a clearance mechanism that would normally remove the protein from brain parenchyma. This study has investigated the capacity of the central nervous system (CNS) phagocytes, microglia cells, to clear exogenous Aβ_1–42 from their environment. Cultured microglia from adult rat CNS have a high capacity to remove Aβ from serum-free medium, shown by immunoblotting experiments. Aβ from incubation medium was attached to the cell surface and could be identified by immunocytochemistry at the light or electron microscopic (EM) level; by EM, Aβ also appeared in phagosome-like intracellular vesicles. Light microscopic immunocytochemistry combined with computer-assisted image analysis showed that cells accumulated Aβ within 24 hr. from culture medium containing from 1 to 20 μg/ml Aβ. Microglial accumulation of Aβ was substantially reduced in the presence of fetal bovine serum. Addition of the protease inhibitor leupeptin to incubation medium with serum resulted in accumulation of Aβ in a membrane-bound intracellular compartment, but not at the cell surface. The increase in intracellular accumulation in the presence of the protease inhibitor indicates a microglial capacity for intracellular degradation of Aβ in the absence of inhibition. The change from predominantly cell-surface accumulation in serum-free medium to predominantly intracellular accumulation with serum may be explained by the presence in serum of carrier proteins that complex with Aβ and target it to cell surface receptors capable of stimulating endocytosis. Microglia were also cultured on unfixed cryostat sections of human brain tissue containing Alzheimer's plaques. Very little Aβ from the tissue was accumulated by the cells, although cultured microglia were found in direct contact with anti-Aβ immunopositive plaques. Possibly Aβ in tissue sections was complexed with other proteins which either inhibited its uptake by microglia or enhanced its proteolysis, preventing cellular accumulation of immunostainable Aβ. The results indicate that cultured microglia effectively remove Aβ from tissue culture medium and from the surface of the dish and concentrate monomer and aggregates of Aβ either on the cell surface or intracellularly. This process may be modified by proteins present in Alzheimer's brain sections. © 1996 Wiley-Liss, Inc.     

α2, α2A, α2B/2C-Adrenoceptor subtype antagonists prevent lipopolysaccharide-induced fever response in rabbits

Exogenous pyrogens, e.g., bacterial lipopolysaccharides (LPS), are thought to stimulate macrophages to release endogenous pyrogens, e.g., TNFα, IL-1 β, and IL-6, which act in the hypothalamus to produce fever. We studied the effect of different α₁⁻ and α₂-adrenoceptor subtype antagonists, applied intraperitoneally, on the febrile response induced by LPS in rabbits. Evidence was obtained that prazosin, an α₁⁻ and α_2B/2C-adrenoceptor antagonist; WB-4101, an α₁⁻ and α_2A-adrenoceptor antagonist; CH-38083, a highly selective α₂-adrenoceptor antagonist (α₂: α₁ > 2000); BRL-44408, an α_2A-adrenoceptor antagonist; and ARC-239, an α_2B/2C⁻ and also α₁-adrenoceptor antagonist, blocked the increase of colonic temperature of the rabbit produced by 2 μg/kg LPS administered intravenously without being able in themselves to affect colonic temperature. In addition, prazosin, WB-4101 and CH-38083 antagonized the fall in skin temperature that occurred at the time when the colonic temperature was rising in control animals injected with LPS. All these results suggest that norepinephrine, through stimulation of both α₁⁻andα₂⁻ (α_2A⁻andα_2B/2C⁻) adrenoceptor subtypes, is involved in producing fever in response to bacterial LPS.     

Differential expression of thymosins β4 and β10 during rat cerebellum postnatal development

The β-thymosins are a family of actin monomer-sequestering proteins widely distributed among vertebrate classes. The most abundant β-thymosins in mammalian species are thymosin β₄ (Tβ₄) and thymosin β₁₀ (Tβ₁₀), two small peptides (43 amino acids) sharing a high degree of sequence homology. In the present work, we have analyzed the distribution of Tβ₄ and Tβ₁₀ in the developing and adult rat cerebellum using in situ hybridization and immunohistochemistry techniques. Our results show that the temporal and cellular patterns of expression of both β-thymosins are different. In the young (7 and 18 postnatal days) and adult (1 and 4 months old) rat cerebellum, Tβ₄ was mainly expressed in the glia (microglia, Golgi epithelial cells and oligodendrocytes), neurons (granule cells and Purkinje cells), and in the capillaries. In 14-month-old rats, the Tβ₄ immunoreactivity was only detected in some microglia cells. In young and adult animals, most of the Tβ₁₀ immunoreactivity was localized in several types of neuronal cells including granule cells, Golgi neurons and Purkinje cells. In old animals, a faint Tβ₁₀ signal could be detected in a few Purkinje cells. Our results suggest that each β-thymosin could play a different function in the control of actin dynamics.     

Cerebrospinal fluid tau, phospho-tau181 and β-amyloid1−42 in idiopathic normal pressure hydrocephalus: a discrimination from Alzheimer's disease

The aim of the present study was the quantitation of total tau protein ( τ _T), tau phosphorylated at threonine 181 ( τ _P-181) and β -amyloid₁₋₄₂ (A β 42) in the cerebrospinal fluid (CSF) of patients with idiopathic normal pressure hydrocephalus (iNPH), Alzheimer's disease (AD) and controls. Double sandwich ELISAs (Innogenetics) were used for the measurements. Total tau was significantly increased in iNPH and highly increased in AD as compared with the control group, whilst A β 42 was decreased in both diseases. CSF τ _P−181 levels were significantly increased only in AD, but not in iNPH as compared with the controls. A cut-off level for τ _T at 300 pg/ml, successfully discriminated AD from normal aging with a 95.8% specificity and 91% sensitivity; whilst the τ _P-181/ τ _T ratio (cut-off value 0.169) was more specific (100%) but less sensitive (92.5%). For the discrimination of iNPH from AD τ _T achieved low specificity (77.8%) but high sensitivity (92.5%), whilst τ _P-181 (cut-off value 47.4) was both sensitive and specific (88.7% and 86.7% respectively) for the discrimination of these disorders. The present study, despite being clinical, supports the notion that CSF τ _P-181 alone or in combination with τ _T may be a useful marker in the discrimination of iNPH from AD.     

Susceptibility and resistance of human autoimmune T cell activation to the immunoregulatory effects of transforming growth factor (TGF) β1, β2 and β1.2

The transforming growth factors type beta (TGF-beta) regulate immune responses by suppressing a variety of leukocyte functions. Using a panel of human autoimmune T cell lines specific for the acetylcholine receptor (AchR) we investigated the immunoregulatory effects of TGF-beta 1, TGF-beta 2, and TGF-beta 1.2. The cytokines have identical effects inhibiting activation of most T line cells and the activation-dependent changes in interleukin-2 (IL-2) receptor and T cell receptor expression. IL-2-dependent growth was not modulated by TGF-beta. However, autoimmune T cell lines specific for AchR differ in their susceptibility to TGF-beta and some are completely refractory. Resistance of autoimmune T cell activation to immunosuppressive cytokines might be an element in the development of chronic autoimmune disease.     

Amyloid β precursor protein-mRNA is expressed throughout cerebral vessel walls

To determine the presence and distribution of cerebrovascular Aβ production we investigated amyloid β precursor protein (AβPP)-mRNA expression by RNA in situ hybridization in patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type, Alzheimer disease and controls. In all subjects, AβPP-mRNA was expressed in endothelial cells, smooth muscle cells, adventitial cells and brain pericytes and/or perivascular cells. Meningeal cells also expressed AβPP-mRNA. AβPP was detected in endothelial cells, smooth muscle cells and adventitial cells. The demonstration of AβPP-mRNA at all vascular sites where amyloid formation can occur supports an important contribution of locally derived Aβ to cerebrovascular amyloidosis.     

Morphological Development of β(1-40) Amyloid Fibrils

The Alzheimer's disease-related peptide β(1-40) amyloid self-associates to form fibrils exhibiting a morphology characteristic of amyloidogenic proteins. The mechanism of this fibrillization process has yet to be fully elucidated. In this study we have immobilized the β(1-40) amyloid to flat gold surfaces using thiol-based self-assembled monolayers. Atomic force microscopy reveals the presence of spherical units of β(1-40) amyloid immediately following the initiation of fibrillization. Short fibrillar structures, termed nascent fibrils, which appear to be formed by the association of these units are also present at this time point. At later time points extended, branching networks of fibrils are observed. Some fibrils exhibit a more beaded appearance and greater axial periodicity than others. No nascent fibrils are seen to be present. We believe that these data identify an early fibril structure which could act as an intermediate in β-amyloid fibrillization. The oligomeric units of which these nascent fibrils are comprised are also determined.     

Amyloid β-protein precursor is associated with extracellular matrix

The ultrastructural distribution of the amyloid beta-protein precursor (ABPP) in cultures of a neuronal cell line is examined. ABPP is associated with the extensive extracellular matrix (ECM) secreted by these cells. The possible role of matrix associated ABPP in the pathogenesis of Alzheimer's disease is discussed.     

Binding and uptake of Aβ1‐42 by primary human astrocytes in vitro

Clearance of the amyloid‐β peptide (Aβ) as a remedy for Alzheimer's disease (AD) is a major target in on‐going clinical trials. In vitro studies confirmed that Aβ is taken up by rodent astrocytes, but knowledge on human astrocyte‐mediated Aβ clearance is sparse. Therefore, by means of flow cytometry and confocal laser scanning microscopy (CLSM), we evaluated the binding and internalization of Aβ1‐42 by primary human fetal astrocytes and adult astrocytes, isolated from nondemented subjects (n = 8) and AD subjects (n = 6). Furthermore, we analyzed whether α1‐antichymotrypsin (ACT), which is found in amyloid plaques and can influence Aβ fibrillogenesis, affects the Aβ uptake by human astrocytes. Upon over night exposure of astrocytes to FAM‐labeled Aβ1‐42 (10 μM) preparations, (80.7 ± 17.7)% fetal and (52.9 ± 20.9)% adult Aβ‐positive astrocytes (P = 0.018) were observed. No significant difference was found in Aβ1‐42 uptake between AD and non‐AD astrocytes, and no influence of ApoE genotype on Aβ1‐42 uptake was observed in any group. There was no difference in the percentage of Aβ‐positive cells upon exposure to Aβ1‐42 (10 μM) combined with ACT (1,000:1, 100:1, and 10:1 molar ratio), versus Aβ1‐42 alone. CLSM revealed binding of Aβ1‐42 to the cellular surfaces and cellular internalization of smaller Aβ1‐42 fragments. Under these conditions, there was no increase in cellular release of the proinflammatory chemokine monocyte‐chemoattractant protein 1, as compared with nontreated control astrocytes. Thus, primary human astrocytes derived from different sources can bind and internalize Aβ1‐42, and fetal astrocytes were more efficient in Aβ1‐42 uptake than adult astrocytes. © 2008 Wiley‐Liss, Inc.     

Cerebrospinal fluid Tau/α‐synuclein ratio in Parkinson's disease and degenerative dementias

Although alpha‐synuclein is the main constituent of Lewy bodies, cerebrospinal fluid determination on its own does not seem fundamental for the diagnosis of synucleinopathies. We evaluated whether the combination of classical biomarkers, Aβ_1–42, total tau, phosphorylated tau, and α‐synuclein can improve discrimination of Parkinson's disease, dementia with Lewy bodies, Alzheimer's disease, and frontotemporal dementia. Aβ_1–42, total tau, phosphorylated tau, and α‐synuclein were measured in a series of patients with Parkinson's disease (n = 38), dementia with Lewy bodies (n = 32), Alzheimer's disease (n = 48), frontotemporal dementia (n = 31), and age‐matched control patients with other neurological diseases (n = 32). Mean α‐synuclein levels in cerebrospinal fluid were significantly lower in the pathological groups than in cognitively healthy subjects. An inverse correlation of α‐synuclein with total tau (r = ?0.196, P < .01) was observed. In the group of patients with Parkinson's disease, Aβ_1–42, total tau, and phosphorylated tau values were similar to controls, whereas total tau/α‐synuclein and phosphorylated tau/α‐synuclein ratios showed the lowest values. Cerebrospinal fluid α‐synuclein alone did not provide relevant information for Parkinson's disease diagnosis, showing low specificity (area under the curve, 0.662; sensitivity, 94%; specificity, 25%). Instead, a better performance was obtained with the total tau/α‐syn ratio (area under the curve, 0.765; sensitivity, 89%; specificity, 61%). Combined determination of α‐synuclein and classical biomarkers in cerebrospinal fluid shows differential patterns in neurodegenerative disorders. In particular, total tau/α‐synuclein and phosphorylated tau/α‐synuclein ratios can contribute to the discrimination of Parkinson's disease. © 2011 Movement Disorder Society