Apamin Improves Prefrontal Nicotinic Impairment in Mouse Model of Alzheimer's Disease.

Disruption of attention is an early and disabling symptom of Alzheimer's disease (AD). The underlying cellular mechanisms are poorly understood and treatment options for patients are limited. These early attention deficits are evident in the TgCRND8 mouse, a well-established murine model of AD that recapitulates several features of the disease. Here, we report severe impairment of the nicotinic receptor-mediated excitation of prefrontal attentional circuitry in TgCRND8 mice relative to wild-type littermate controls. We demonstrate that this impairment can be remedied by apamin, a bee venom neurotoxin peptide that acts as a selective antagonist to the SK family of calcium-sensitive potassium channels. We probe this seeming upregulation of calcium-sensitive inhibition and find that the attenuated nicotinic firing rates in TgCRND8 attention circuits are mediated neither by greater cellular calcium signals nor by elevated SK channel expression. Instead, we find that TgCRND8 mice show enhanced functional coupling of nicotinic calcium signals to inhibition. This SK-mediated inhibition exerts a powerful negative feedback on nicotinic excitation, dampening attention-relevant signaling in the TgCRND8 brain. These mechanistic findings identify a new cellular target involved in the modulation of attention and a novel therapeutic target for early attention deficits in AD.

Venular amyloid accumulation in transgenic Fischer 344 Alzheimer's disease rats.

Strong evidence demonstrates a significant association between cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD). For this reason, interest in understanding the underlying vascular pathologies that contribute to AD remain. CAA research has primarily focused on arterioles and capillaries, overlooking the draining venules. Therefore, this study sought to examine venular amyloid pathology and its relationship to arteriolar amyloidosis throughout AD progression in the TgF344-AD rat model. Antibodies targeting the amyloid-beta peptide (Aß) sequence suggest morphological differences between arteriolar and venular amyloid. Mass spectrometric analyses of isolated cortical parenchymal plaques, arteriolar and venular amyloid demonstrated presence of Aß in all three samples, as well as proteins known to be associated with AD. Histopathological analysis indicates a significant age effect for both arteriolar and venular amyloid accumulation, with accumulation initiated in the somatosensory cortex followed by the motor and cingulate cortex. Lastly, significant arteriolar amyloid accumulates relative to venular amyloid deposition in AD progression. Overall, understanding venular and arteriolar amyloid pathology provides insight into the complex connection between CAA and AD.

Therapeutically effective antibodies against amyloid-beta peptide target amyloid-beta residues 4-10 and inhibit cytotoxicity and fibrillogenesis.

Immunization of transgenic mouse models of Alzheimer disease using amyloid-beta peptide (Abeta) reduces both the Alzheimer disease-like neuropathology and the spatial memory impairments of these mice. However, a therapeutic trial of immunization with Abeta42 in humans was discontinued because a few patients developed significant meningo-encephalitic cellular inflammatory reactions. Here we show that beneficial effects in mice arise from antibodies selectively directed against residues 4-10 of Abeta42, and that these antibodies inhibit both Abeta fibrillogenesis and cytotoxicity without eliciting an inflammatory response. These findings provide the basis for improved immunization antigens as well as attempts to design small-molecule mimics as alternative therapies.

Utilizing supervised machine learning to identify microglia and astrocytes in situ: implications for large-scale image analysis and quantification.

BACKGROUND: The evaluation of histological tissue samples plays a crucial role in deciphering preclinical disease and injury mechanisms. High-resolution images can be obtained quickly however data acquisition are often bottlenecked by manual analysis methodologies. NEW METHOD: We describe and validate a pipeline for a novel machine learning-based analytical method, using the Opera High-Content Screening system and Harmony software, allowing for detailed image analysis of cellular markers in histological samples. RESULTS: To validate the machine learning pipeline, analyses of single proteins in mouse brain sections were utilized. To demonstrate adaptability of the pipeline for multiple cell types and epitopes, the percent brain coverage of microglial cells, identified by ionized calcium binding adaptors molecule 1 (Iba1), and of astrocytes, by glial fibrillary acidic protein (GFAP) demonstrated no significant differences between automated and manual analyses protocols. Further to examine the robustness of this protocol for multiple proteins simultaneously labeling of rat brain sections were utilized; co-localization of astrocytic endfeet on blood vessels, using aquaporin-4 and tomato lectin respectively, were efficiently identified and quantified by the novel pipeline and were not significantly different between the two analyses protocols. Comparison with Existing Methods: The automated platform maintained the sensitivity and accuracy of manual analysis, while accomplishing the analyses in 1/200th of the time. CONCLUSIONS: We demonstrate the benefits and potential of adapting an automated high-throughput machine-learning analytical approach for the analysis ofin situ tissue samples, show effectiveness across different animal models, while reducing analysis time and increasing productivity.

Phosphatidylinositol and inositol involvement in Alzheimer amyloid-beta fibril growth and arrest.

A key pathological feature of Alzheimer's disease is the formation and accumulation of amyloid fibres. The major component is the 39 to 42 residue amyloid-beta peptide (Abeta) which is an internal proteolytic fragment of the integral membrane amyloid precursor protein. Aggregation of Abeta into insoluble amyloid fibres is a nucleation-dependent event that may be modulated by the presence of amyloid-associated molecules. Fibril formation is also associated with neurotoxicity which may be the result of specific Abeta interactions with membrane proteins and/or lipids. Using circular dichroism spectroscopy, tyrosine fluorescence spectroscopy and electron microscopy, we have examined the binding of Abeta peptides 1-40 (Abeta40) and 1-42 (Abeta42) to the glycolipid, phosphatidylinositol (PI), and different inositol headgroups. At pH 6.0 and in the presence of PI vesicles, both Abeta40 and Abeta42 adopted an amyloidogenic beta-structure. In contrast, at neutral pH only Abeta42 folded into a beta-structure in the presence of PI vesicles. To determine whether the induction of beta-structure stemmed from interactions with the headgroup of PI, the effects of inositol derivatives on Abeta were also examined. At pH 7.0, myo-inositol was sufficient to induce beta-structure in Abeta42 but had no effect on the conformation of Abeta40. Myo-inositol may promote beta-structure as a result of its ability to be both a hydrogen-bond donor and acceptor. Mono-, di- and triphosphorylated forms of inositol had reduced ability to induce beta-structure in both peptides. The results from this study indicate that interaction of Abeta40 and Abeta42 with PI acts as a seed for fibril formation while myo-inositol stabilizes a soluble Abeta42 micelle.

Cholesterol, a modulator of membrane-associated Abeta-fibrillogenesis and neurotoxicity.

Recent studies have suggested that cholesterol, an important determinant of the physical state of biological membranes, plays a significant role in the development of Alzheimer's disease. We have employed in situ scanning probe microscopy, fluorescence anisotropy, and electron microscopy to investigate how cholesterol levels within total brain lipid bilayers effect amyloid beta-peptide (Abeta)-assembly. Fluorescence anisotropy measurements revealed that the relative fluidity of the total brain lipid membranes was influenced by the level of cholesterol and the addition of Abeta40 resulted in a decrease in the overall vesicle fluidity. In situ scanning probe microscopy performed on supported planar bilayers of total brain lipid revealed a correlation between membrane fluidity, as influenced by cholesterol level, and the extent of Abeta-insertion and subsequent fibrillogenesis. These observations were consistent with fluorescence microscopy studies of PC-12 and SH-SY5Y cell lines exposed to exogenous Abeta, which revealed an inverse correlation between membrane cholesterol level, and Abeta-cell surface binding and subsequent cell death. These results collectively suggest that Abeta-cell surface interactions are mediated by cellular cholesterol levels, the distribution of cholesterol throughout the cell, and membrane fluidity.

Amyloid beta-protein (A beta) associated with lipid molecules: immunoreactivity distinct from that of soluble A beta.

We previously identified a novel amyloid beta-protein (A beta), that binds to GM1 ganglioside, in brains exhibiting the early pathological changes of AD. In this study, we raised monoclonal antibodies, using membrane fractions containing abundant GM1 ganglioside-bound A beta as antigens. Monoclonal antibody 4396, produced in this study, immunoprecipitates A beta42 in the membrane fractions of brains with diffuse plaques, but does not react with soluble A beta42 or GM1 ganglioside. Furthermore, this antibody recognizes the A beta bound to lipid vesicles containing GM1 ganglioside, and unexpectedly, phosphatidylinositol. In contrast, a control anti-A beta monoclonal antibody does not recognize the A beta bound to these lipid vesicles. These results indicate that A beta associated with lipids has an immunoreactivity distinct from that of soluble A.

Augmentation of pulmonary reactions to quartz inhalation by trace amounts of iron-containing particles.

Fracturing quartz produces silica-based radicals on the fracture planes and generates hydroxyl radicals (.OH) in aqueous media. .OH production has been shown to be directly associated with quartz-induced cell damage and phagocyte activation in vitro. This .OH production in vitro is inhibited by desferrioxamine mesylate, an Fe chelator, indicating involvement of a Fenton-like reaction. Our objective was to determine if Fe contamination increased the ability of inhaled quartz to cause inflammation and lung injury. Male Fischer 344 rats were exposed 5 hr/day for 10 days to filtered air, 20 mg/m3 freshly milled quartz (57 ppm Fe), or 20 mg/m3 freshly milled quartz contaminated with Fe (430 ppm Fe). High Fe contamination of quartz produced approximately 57% more reactive species in water than quartz with low Fe contamination. Compared to inhalation of quartz with low Fe contamination, high Fe contamination of quartz resulted in increases in the following responses: leukocyte recruitment (537%), lavageable red blood cells (157%), macrophage production of oxygen radicals measured by electron spin resonance or chemiluminescence (32 or 90%, respectively), nitric oxide production by macrophages (71%), and lipid peroxidation of lung tissue (38%). These results suggest that inhalation of freshly fractured quartz contaminated with trace levels of Fe may be more pathogenic than inhalation of quartz alone.

The Neu-Laxova syndrome in female sibs: clinical and pathological features with prenatal diagnosis in the second sib.

We report on affected sisters with the Neu-Laxova syndrome. Prenatal diagnosis of the condition was achieved by serial ultrasound examinations which demonstrated abnormal fetal growth in the second affected fetus before 24 weeks gestation.

A sulfated proteoglycan aggregation factor mediates amyloid-beta peptide fibril formation and neurotoxicity.

Proteoglycans are associated with senile plaques in Alzheimer's disease and may be involved in A beta fibril formation and plaque formation. In vitro, glycosaminoglycans have been shown to inhibit the proteolysis of A beta fibrils, accelerate formation and maintain their stability. To model their interaction, we investigated the binding of a sulfated proteoglycan derived from a natural source; marine sponge Microciona prolifera aggregation factor (MAF). This species-specific re-aggregation of sponge cells has two functional properties, a Ca2+ independent cell binding activity and a Ca2+ dependent self-aggregation. It has been shown that a novel sulfated disaccharide and a pyruvylated trisaccharide are important in the Ca(2+)-dependent MAF aggregation. Aggregation demonstrated by homophilic binding of MAF subunits may be chemically distinct from other heterotypic binding effects. We investigated A beta-MAF interactions and show that MAF induces a structural transition in A beta 40 and A beta 42 from random to beta-structure as detected by circular dichroism spectroscopy. Electron microscopy revealed that the structural transition correlated with an increase in the number of A beta 40 and A beta 42 aggregated that have a truncated fibrillar morphology. Finally, MAF increased A beta-induced toxicity of nerve growth factor (NGF)-differentiated PC-12 cells in the absence of Ca2+. The addition of Ca2+ to MAF-A beta incubations resulted in a moderate attenuation of toxicity possibly due to a reduction in A beta-cell interactions caused by extensive lateral aggregation of the MAF-A beta complexes. Our results indicate that A beta is generally susceptible to proteoglycan-mediated aggregation and fibril formation. We also propose that the MAF model system may be useful in delineating these interactions and represent a means to develop and examine potential inhibitors of the proteoglycan effects.

Assembly of Alzheimer's amyloid-beta fibrils and approaches for therapeutic intervention.

Amyloid plaques are the principal features of Alzheimers disease (AD) pathology and are considered to be a major factor in the disease process. These fibrillar deposits are composed primarily of the 40-42 residue amyloid-beta (Abeta) peptide which is a proteolytic product of a larger membrane precursor protein. Electron microscopy and X-ray diffraction have revealed that the mature amyloid fibrils are assembled as a highly beta-sheet polymer that has a well-defined protofilament quaternary structure. This organization is observed for amyloid fibrils from a wide variety of disorders and appears to represent a structural superfamily. Amyloid plaques also contain a number of other components such as proteoglycans that contain highly sulfated glycosaminoglycan (GAG) chains. These amyloid-associated elements may contribute to the aggregation and/or stabilization of Abeta as insoluble fibrils. We have recently developed an aggressive model for Abeta plaque formation in transgenic mice that exhibits an "early-onset" phenotype. Immunocytochemistry has demonstrated that even with this rapid progression, Abeta deposits within the neuropil and cerebrovascular system all co-localize with heparan sulfate proteoglycans (HSPG). These findings indicate a number of structural features that can be targeted as potential sites for the development of amyloid inhibitors. In addition, the use of small compounds that interfere with the proteoglycan-amyloid pathway may be effective therapeutic agents that can be assessed through the use of these transgenic models.

Cholesterol, a modulator of membrane-associated Abeta-fibrillogenesis.

One of the major pathological features of Alzheimer's disease (AD) is the presence of extracellular amyloid plaques that are predominantly composed of the amyloid-beta peptide (Abeta). Characterization of plaques demonstrated the predominance of two peptides differing at the carboxyl terminus by two hydrophobic amino acids, Abeta40 and Abeta42. Diffuse plaques associated with AD are composed predominantly of Abeta42, whereas senile plaques contain both Abeta40 and Abeta42. Recently, it has been suggested that diffuse plaque formation is initiated as a plasma membrane-bound Abeta species and that Abeta42 is the critical component. In order to investigate this hypothesis, we have examined Abeta40/42-lipid interactions using in situ atomic force microscopy, electron microscopy, and fluorescence anisotropy. While the association of Abeta42 with planar bilayers resulted in peptide aggregation, but no fiber formation, this was not the case for Abeta40, where we observed preferential fiber formation. Cholesterol, a key membrane component and modulating factor in AD, is inversely correlated with the extent of Abeta40/42-bilayer interaction. These results were confirmed using fluorescence anisotropy to evaluate the effect of Abeta on membrane fluidity and fluorimetry to confirm membrane integrity. Our results suggest that the enhanced amyloidogenic properties of Abeta42 are not correlated with fibril formation, but with aggregation on bilayer surfaces.

Neuropathological, biochemical and genetic alterations in AD.

The molecular and cellular processes that lead to the production of the amyloid beta (A beta) peptide and some of the processes associated with A beta fibrillogenesis and neurotoxicity have recently been elucidated. Experimental results have suggested that abnormalities in the processing of the beta-amyloid precursor protein (beta APP) are central to the pathogenesis of Alzheimer's disease (AD). beta APP processing includes two mutually exclusive proteolytic cleavage pathways, one involving the putative gamma-secretase enzyme, the identity of which remains unknown. Recent evidence has suggested the presenilin 1 and presenilin 2 genes are necessary for gamma-secretase activities. Another gene associated with susceptibility to AD is the apolipoprotein E (APOE) gene. Given the important role that abnormal processing of beta APP plays in the genesis of AD, most current efforts are directed at either modulating A beta peptide production or inhibiting its ability to aggregate into fibrils and cause neurotoxicity. To inhibit A beta production, one strategy might be to inhibit either beta-secretase or gamma-secretase. Several approaches to the inhibition of A beta aggregation are under investigation.

Phenotypic and cell cycle properties of human oligodendrocytes in vitro.

The remyelination, albeit limited, which occurs at the lesion sites in the central nervous in multiple sclerosis has been attributed to both myelin production by previously myelinating cells and to precursor cells which mature into myelin-producing cells. Oligodendrocyte (OL) number may be increased at the periphery of the lesions. In this study, we assessed the state of maturation and cell cycle-dependent properties of OLs derived from surgically resected adult human cerebral cortex specimens. In 6-day-old OL cultures, a small proportion of cells (14.1 +/- 3.5%: range 4-24%) expressed an immature phenotype, defined as A007+:myelin basic protein (MBP)-negative. Using lack of statin expression as an index of cells exiting the G0 phase of the cell cycle, we observed that 4.6 +/- 1.6% of A007+ cells, but only rare MBP+ cells (0.4 +/- 1.8%) were non-reactive with the anti-statin antibody, S44. The proportion of non-statin-reactive cells was not affected by treatment with basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) or insulin-like growth factor (IGF). The oligodendrocytes did not incorporate BrdU during a 48-h pulse and did not immunoreact with Ki-67 antibody. In 4-week-old cultures, we found that all A007+ cells were also MBP+ and that 99.5 +/- 0.7% were statin-positive. Exposing 4-week-old OLs to conditions of serum deprivation or to 1,000 units/ml of recombinant human TNF-beta for 4 days induced nuclear fragmentation in a high proportion (> 70%) of cells, as measured by a TUNEL technique; in these cultures, a similarly high proportion of cells were non-immunoreactive with anti-statin antibody. Our results suggest that a small number of phenotypic 'pre-oligodendrocytes' can be derived from the adult human CNS and that a proportion of these cells have exited the G0 phase of the cell cycle. Attempt at cell cycling, however, could reflect abortive mitosis and activation of programmed cell death.

Effect of tumor necrosis factor alpha and beta on human oligodendrocytes and neurons in culture.

Cytokines produced by infiltrating hematogenous cells or by glial cells activated during the course of central nervous system disease or trauma are implicated as mediators of tissue injury. In this study, we have assessed the extent and mechanism of injury of human-derived CNS oligodendrocytes and neurons in vitro mediated by the cytokines tumor necrosis factor alpha and beta and compared these with the tumor necrosis factor independent effects mediated by activated CD4+ T-cells. We found that activated CD4+ T-cells, but not tumor necrosis factor alpha or beta, could induce significant release of lactate dehydrogenase, a measure of cell membrane lysis, from oligodendrocytes within 24 hr. Neither induced DNA fragmentation as measured using a fluorescence nick-end labelling technique. After a more prolonged time period (96 hr), tumor necrosis factor alpha did induce nuclear fragmentation changes in a significant proportion of oligodendrocytes without increased lactate dehydrogenase release. The extent of DNA fragmentation was comparable to that induced by serum deprivation. Tumor necrosis factor beta effects were even more pronounced. In contrast to oligodendrocytes, the extent of DNA fragmentation, assessed by propidium iodide staining, induced in neurons by tumor necrosis factor alpha was less than that induced by serum deprivation. In-situ hybridization studies of human adult glial cells in culture indicated that astrocytes, as well as microglia, can express tumor necrosis factor alpha mRNA.

Reactivity of two anti-galactosyl ceramide antibodies towards myelin basic protein.

Two anti-galactosyl ceramide antibodies (polyclonal Ab142 and a monoclonal antibody) were characterized in terms of their reactivity towards purified lipids and myelin basic protein. Polyclonal Ab142 is a rabbit anti-mouse galactosyl ceramide (Gal C) IgG. Antigenic recognition is dependent on both galactose and ceramide since neither could inhibit galactosyl ceramide binding by more than 10%. MAb-Gal C is a monoclonal antibody raised in mice against galactosyl ceramide. Binding of MAb-Gal C to Gal-C was equally inhibited by ceramide and galactose to approximately 50%, indicating that both groups are important for antibody recognition. MAb-Gal C was also shown to be reactive with the structurally related lipids, sphingomyelin and sulfatide. Polyclonal Ab142, although raised against Gal C, was shown to be 3-fold more reactive with component 8 (C-8) of myelin basic protein than Gal C. On the other hand, the MAb-Gal C which also reacted with C-8 was 2-fold less reactive with C-8 than with Gal C. Neither of these antibodies were reactive with component 1 (C-1) of myelin basic protein. An anti-MBP IgG was shown to be reactive with C-1 and C-8 but unreactive with Gal C. In competitive inhibition ELISA, C-8 was able to compete out 44% and 41% of Gal C binding to polyclonal Ab142 and MAb, respectively. The reverse competition demonstrated that Gal C could inhibit 75% of C-8 binding to both antibodies. D-galactose was unable to inhibit C-8 binding to either antibody, whereas ceramide was as efficient as Gal C.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactivity of normal T-cell lines to MBP isolated from normal and multiple sclerosis white matter.

T-cell reactivity to human myelin basic protein (MBP) has been extensively studied using T-cell lines and clones generated from both peripheral blood and cerebrospinal fluid, from normal controls and multiple sclerosis (MS) patients. These studies have largely utilized myelin basic protein isolated from control human adult white matter. In our study, we used MBP reactive T-cell lines as a probe to investigate antigenic differences in a series of MBP preparations isolated from either control human white matter or white matter from the central nervous system (CNS) of MS patients. Autologous peripheral blood derived mononuclear cells were used as antigen presenting cells (APC). Although the majority of T-cells were found to react equally well with all preparations of MBP isolated from both control and MS white matter, we were also able to identify T-cell lines which reacted well with all preparations of MBP isolated from controls but failed to react with MBP isolated from MS white matter. These differences were unlikely to reflect differences in degradation products or excess peptides present in the MS brain since SDS-PAGE and HPLC did not show any difference in the MS samples compared to the controls, and the concentration response curves for a human T-cell clone specific for the 84-102 region of MBP were similar for all the MBP preparations. We did not detect differences in amino acid content amongst MBP preparations although single amino acid substitutions cannot be ruled out. These results raise the possibility that MBP isolated from MS brain may differ in charge microheterogeneity which would affect antigenic determinants.(ABSTRACT TRUNCATED AT 250 WORDS)

Oligodendrocytes and myelin.

Although it was initially thought to be a passive structure, the oligodendrocyte and myelin unit is now considered highly dynamic with many important functions beyond its role in myelination. Inhibitors of neurite growth can be localized to oligodendrocyte and myelin, highlighting the importance of oligodendrocytes in general CNS maintenance and in neuronal regeneration following many types of CNS insults. Understanding of the genesis of oligodendrocytes and myelin is increasing and will surely lead to better strategies to treat demyelinating diseases including MS.

Time course of pulmonary response of rats to inhalation of crystalline silica: histological results and biochemical indices of damage, lipidosis, and fibrosis.

Previous studies have determined that alpha-quartz (crystalline silica) can cause pulmonary inflammation, damage, and fibrosis. However, the temporal relationship between silica inhalation and pulmonary inflammation, damage, and fibrosis has not been fully examined. To address this gap in our knowledge of silica-induced pulmonary fibrosis, a chronic inhalation study using rats was designed. Specifically, rats were exposed to a silica aerosol (15 mg/m3 silica, 6 h/d, 5 d/wk, 116 d), and measurements of pulmonary inflammation, damage, and fibrosis were monitored throughout the study. We report (1) data demonstrating that the silica aerosol generation and exposure system produced a consistent silica aerosol of respirable size particles; (2) the time course of silica deposition in the lung; (3) calculations that demonstrate that the rats were not in pulmonary overload; (4) histopathological data demonstrating time-dependent enhancement of silica-induced alveolitis, epithelial hypertrophy and hyperplasia, alveolar lipoproteinosis, and pulmonary fibrosis in the absence of overload; and (5) biochemical data documenting the development of lipidosis, lung damage, and fibrosis.

Particle activity and in vivo pulmonary response to freshly milled and aged alpha-quartz.

This study examined the possibility of freshly fractured alpha-quartz being more toxic and inflammatory in vivo than aged quartz of the same composition and particle size. Fresh quartz was generated by a jet mill, and used immediately, while aged dust was stored for two months before use. Both the production of hydrogen peroxide and hydroxyl radicals and the analysis of surface radicals verified the enhanced surface activity of fresh quartz. Male Fischer 344 rats were exposed to fresh or aged alpha-quartz by inhalation (20 mg center dot m-3, 5 h per day, 5 d per week, for 2 weeks) and their pulmonary responses were determined 1--3 d postexposure. Exposure to aged quartz resulted in an increase in cytotoxic and inflammatory parameters. In comparison, the inhalation of freshly cleaved quartz resulted in dramatically greater increases in all of the pulmonary responses. This finding suggests that exposure to freshly machined quartz may result in a greater risk of pulmonary disease.