Modulation of amyloid-ß aggregation by metal complexes with a dual binding mode and their delivery across the blood-brain barrier using focused ultrasound.

One of the key hallmarks of Alzheimer's disease is the aggregation of the amyloid-ß peptide to form fibrils. Consequently, there has been great interest in studying molecules that can disrupt amyloid-ß aggregation. While a handful of molecules have been shown to inhibit amyloid-ß aggregation in vitro, there remains a lack of in vivo data reported due to their inability to cross the blood-brain barrier. Here, we investigate a series of new metal complexes for their ability to inhibit amyloid-ß aggregation in vitro. We demonstrate that octahedral cobalt complexes with polyaromatic ligands have high inhibitory activity thanks to their dual binding mode involving π-π stacking and metal coordination to amyloid-ß (confirmed via a range of spectroscopic and biophysical techniques). In addition to their high activity, these complexes are not cytotoxic to human neuroblastoma cells. Finally, we report for the first time that these metal complexes can be safely delivered across the blood-brain barrier to specific locations in the brains of mice using focused ultrasound.

Annexin A1 restores cerebrovascular integrity concomitant with reduced amyloid-ß and tau pathology.

Alzheimer's disease, characterized by brain deposits of amyloid-ß plaques and neurofibrillary tangles, is also linked to neurovascular dysfunction and blood-brain barrier breakdown, affecting the passage of substances into and out of the brain. We hypothesized that treatment of neurovascular alterations could be beneficial in Alzheimer's disease. Annexin A1 (ANXA1) is a mediator of glucocorticoid anti-inflammatory action that can suppress microglial activation and reduce blood-brain barrier leakage. We have reported recently that treatment with recombinant human ANXA1 (hrANXA1) reduced amyloid-ß levels by increased degradation in neuroblastoma cells and phagocytosis by microglia. Here, we show the beneficial effects of hrANXA1 in vivo by restoring efficient blood-brain barrier function and decreasing amyloid-ß and tau pathology in 5xFAD mice and Tau-P301L mice. We demonstrate that young 5xFAD mice already suffer cerebrovascular damage, while acute pre-administration of hrANXA1 rescued the vascular defects. Interestingly, the ameliorated blood-brain barrier permeability in young 5xFAD mice by hrANXA1 correlated with reduced brain amyloid-ß load, due to increased clearance and degradation of amyloid-ß by insulin degrading enzyme (IDE). The systemic anti-inflammatory properties of hrANXA1 were also observed in 5xFAD mice, increasing IL-10 and reducing TNF-α expression. Additionally, the prolonged treatment with hrANXA1 reduced the memory deficits and increased synaptic density in young 5xFAD mice. Similarly, in Tau-P301L mice, acute hrANXA1 administration restored vascular architecture integrity, affecting the distribution of tight junctions, and reduced tau phosphorylation. The combined data support the hypothesis that blood-brain barrier breakdown early in Alzheimer's disease can be restored by hrANXA1 as a potential therapeutic approach.

Transfer of SAR information from hypotensive indazole to indole derivatives acting at α-adrenergic receptors: In vitro and in vivo studies.

In a search for novel antihypertensive drugs we applied scaffold hopping from the previously described α1-adrenergic receptor antagonists, 1-[(imidazolin-2-yl)methyl]indazoles. The aim was to investigate whether the α-adrenergic properties of the indazole core were transferable to the indole core. The newly obtained 1-[(imidazolin-2-yl)methyl]indole analogues were screened in vitro for their binding affinities for α1-and α2-adrenoceptors, which allowed the identification of the target-based SAR transfer (T_SAR transfer) as well as structure-based SAR transfer (S_SAR transfer) events. However, when screened in vivo with use of anaesthetized male Wistar rats, the new indole ligands showed a different hemodynamic profile than expected. Instead of the immediate hypotensive effect characteristic of peripheral vasodilatator α1 blockers, a biphasic effect was observed, reminiscent of clonidine-like centrally acting antihypertensive agents. This was supported by subsequent in vitro functional studies in [(35)S]GTPγS binding assay, where the indole analogues displayed partial agonist properties at α2-adrenergic receptors. Since no correlation was found between the in vitro binding to α-adrenoceptors and the in vivo hemodynamic effects of the two series of indazole and indole bioisosteric compounds, in a search for new imidazoline-containing adrenergic drugs, the structure-based SAR transfer information obtained from in vitro binding studies should be treated with caution.

Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke.

Ischaemic stroke is caused by occlusive thrombi in the cerebral vasculature. Although tissue-plasminogen activator (tPA) can be administered as thrombolytic therapy, it has major limitations, which include disruption of the blood-brain barrier and an increased risk of bleeding. Treatments that prevent or limit such deleterious effects could be of major clinical importance. Activated protein C (APC) is a natural anticoagulant that regulates thrombin generation, but also confers endothelial cytoprotective effects and improved endothelial barrier function mediated through its cell signalling properties. In murine models of stroke, although APC can limit the deleterious effects of tPA due to its cell signalling function, its anticoagulant actions can further elevate the risk of bleeding. Thus, APC variants such as APC(5A), APC(Ca-ins) and APC(36-39) with reduced anticoagulant, but normal signalling function may have therapeutic benefit. Human and murine protein C (5A), (Ca-ins) and (36-39) variants were expressed and characterised. All protein C variants were secreted normally, but 5-20% of the protein C (Ca-ins) variants were secreted as disulphide-linked dimers. Thrombin generation assays suggested reductions in anticoagulant function of 50- to 57-fold for APC(36-39), 22- to 27-fold for APC(Ca-ins) and 14- to 17-fold for APC(5A). Interestingly, whereas human wt APC, APC(36-39) and APC(Ca-ins) were inhibited similarly by protein C inhibitor (t½ - 33 to 39 mins), APC(5A) was inactivated ~9-fold faster (t½ - 4 mins). Using the murine middle cerebral artery occlusion ischaemia/repurfusion injury model, in combination with tPA, APC(36-39), which cannot be enhanced by its cofactor protein S, significantly improved neurological scores, reduced cerebral infarct area by ~50% and reduced oedema ratio. APC(36-39) also significantly reduced bleeding in the brain induced by administration of tPA, whereas wt APC did not. If our data can be extrapolated to clinical settings, then APC(36-39) could represent a feasible adjunctive therapy for ischaemic stroke.

Xenon Treatment Protects against Remote Lung Injury after Kidney Transplantation in Rats.

BACKGROUND: Ischemia-reperfusion injury (IRI) of renal grafts may cause remote organ injury including lungs. The authors aimed to evaluate the protective effect of xenon exposure against remote lung injury due to renal graft IRI in a rat renal transplantation model. METHODS: For in vitro studies, human lung epithelial cell A549 was challenged with H2O2, tumor necrosis factor-α, or conditioned medium from human kidney proximal tubular cells (HK-2) after hypothermia-hypoxia insults. For in vivo studies, the Lewis renal graft was stored in 4°C Soltran preserving solution for 24 h and transplanted into the Lewis recipient, and the lungs were harvested 24 h after grafting. Cultured lung cells or the recipient after engraftment was exposed to 70% Xe or N2. Phospho (p)-mammalian target of rapamycin (mTOR), hypoxia-inducible factor-1α (HIF-1α), Bcl-2, high-mobility group protein-1 (HMGB-1), TLR-4, and nuclear factor κB (NF-κB) expression, lung inflammation, and cell injuries were assessed. RESULTS: Recipients receiving ischemic renal grafts developed pulmonary injury. Xenon treatment enhanced HIF-1α, which attenuated HMGB-1 translocation and NF-κB activation in A549 cells with oxidative and inflammatory stress. Xenon treatment enhanced p-mTOR, HIF-1α, and Bcl-2 expression and, in turn, promoted cell proliferation in the lung. Upon grafting, HMGB-1 translocation from lung epithelial nuclei was reduced; the TLR-4/NF-κB pathway was suppressed by xenon treatment; and subsequent tissue injury score (nitrogen vs. xenon: 26 ± 1.8 vs. 10.7 ± 2.6; n = 6) was significantly reduced. CONCLUSION: Xenon treatment confers protection against distant lung injury triggered by renal graft IRI, which is likely through the activation of mTOR-HIF-1α pathway and suppression of the HMGB-1 translocation from nuclei to cytoplasm.

The impact of surgery and anesthesia on post-operative cognitive decline and Alzheimer's disease development: biomarkers and preventive strategies.

Alzheimer's disease (AD) is a major social and clinical burden in the elderly, affecting 5% of people aged over 65 and 20% aged over 80. Despite improved management, a cure has not been found and hence analysis of predisposing factors to identify preventive strategies has become increasingly important. Surgery and anesthesia have been proposed to increase the incidence of post-operative cognitive decline (POCD) and AD. This is hypothesized to be the result of a malignant neuroinflammatory response and subsequent synaptic impairment in the elderly and susceptible individuals. As a result, strategies are being explored to prevent surgery and anesthesia induced cognitive impairment. Whereas previously the diagnosis of AD was primarily dependent on clinical examination, biomarkers such as inflammatory cytokines, amyloid-ß, and tau deposition in the cerebrospinal fluid have received increased attention. Nonetheless, AD is currently still treated symptomatically with acetylcholinesterase inhibitors and NMDA antagonists to improve cholinergic transmission and prevent glutamatergic excitotoxicity. Therapeutic success is, however, often not achieved, since these treatment methods do not address the ongoing neuroinflammatory processes and hence novel therapeutic and protective strategies are urgently needed. This review provides an insight into the current understanding of age-related cognitive impairment post-surgery and reflects on novel markers of AD pathogeneses exploring their use as targets for treatment. It gives a summary of recent efforts in preventing and treating POCD or AD with regards to the choice and depth of anesthesia, surgical strategy, and peri-operative medication, and discusses the mechanism of action and therapeutic prospects of novel agents.

The therapeutic potential of atorvastatin in a mouse model of postoperative cognitive decline.

OBJECTIVE: Postoperative cognitive decline is emerging as a significant complication of surgery among older adults. Animal models indicate a central role of hippocampal inflammatory responses in the pathophysiology of postoperative cognitive decline. We hypothesized that atorvastatin, shown to exert neuroprotective potential in central nervous system (CNS) disorders, would attenuate neuroinflammation and improve cognitive function in mice after surgery and anesthesia. METHODS: C57BL6 adult mice were pretreated with atorvastatin (250 µg) or vehicle, orally, for 5 days before undergoing unilateral nephrectomy under isoflurane anesthesia. We evaluated behavioral parameters related to cognitive function (fear conditioning and Morris Water Maze) and determined systemic and hippocampal interleukin-1ß levels, postoperatively. Endothelial COX-2 expression, gross NF-κB and microglial (IBA1, CD68) activation, synaptic function (synapsin-1, PSD95, COX-2), heme oxygenase-1, and GSK3ß were also examined. RESULTS: Surgery induced a significant reduction in hippocampal-dependent fear response that was attenuated by treatment with atorvastatin, which also preserved spatial memory on day 7 after surgery. Atorvastatin evoked significant protection from hippocampal interleukin-1ß production, but not systemic interleukin-1ß production, accompanied by a marked reduction in hippocampal endothelial COX-2, NF-κB activation and decreased microglial reactivity. Surgery triggered an acute decline in synapsin-1, paralleled by an increase in postsynaptic COX-2 that was partially attenuated by atorvastatin. Furthermore, phosphorylation and inactivation of neuronal GSK3ß was significantly enhanced after atorvastatin treatment. CONCLUSIONS: These findings indicate that cognitive decline is very likely associated with synaptic pathology after systemic and central inflammation induced by peripheral surgery/isoflurane anesthesia and suggest that the anti-inflammatory and neuroprotective properties of atorvastatin provide a rationale for its use as a therapeutic strategy for postoperative cognitive decline.

Isoflurane, a commonly used volatile anesthetic, enhances renal cancer growth and malignant potential via the hypoxia-inducible factor cellular signaling pathway in vitro.

BACKGROUND: Growing evidence indicates that perioperative factors, including choice of anesthetic, affect cancer recurrence after surgery although little is known about the effect of anesthetics on cancer cells themselves. Certain anesthetics are known to affect hypoxia cell signaling mechanisms in healthy cells by up-regulating hypoxia-inducible factors (HIFs). HIFs are also heavily implicated in tumorigenesis and high levels correlate with poor prognosis. METHODS: Renal cell carcinoma (RCC4) cells were exposed to isoflurane for 2 h at various concentrations (0.5-2%). HIF-1α, HIF-2α, phospho-Akt, and vascular endothelial growth factor A levels were measured by immunoblotting at various time points (0-24 h). Cell migration was measured across various components of extracellular matrix, and immunocytochemistry was used to analyze proliferation rate and cytoskeletal changes. RESULTS: Isoflurane up-regulated levels of HIF-1α and HIF-2α and intensified expression of vascular endothelial growth factor A. Exposed cultures contained significantly more cells (1.81 ± 0.25 vs. 1.00 of control; P = 0.03) and actively proliferating cells (89.4 ± 2.80 vs. 64.74 ± 7.09% of control; P = 0.016) than controls. These effects were abrogated when cells were pretreated with the Akt inhibitor, LY294002. Exposed cells also exhibited greater migration on tissue culture-coated (F = 16.89; P = 0.0008), collagen-coated (F = 20.99; P = 0.0003), and fibronectin-coated wells (F = 8.21; P = 0.011) as along with dramatic cytoskeletal rearrangement, with changes to both filamentous actin and α-tubulin. CONCLUSIONS: These results provide evidence that a frequently used anesthetic can exert a protumorigenic effect on a human cancer cell line. This may represent an important contributory factor to high recurrence rates observed after surgery.

N-(Imidazolidin-2-ylidene)-1-arylmethanamine oxides: synthesis, structure and pharmacological evaluation.

A high yielding three-step procedure was applied for the synthesis of N-(imidazolidin-2-ylidene)-1-arylmethanamine oxides 3 (α-aminonitrones) starting from the easily accessible imidazolidin-2-one O-benzyl oxime 1. The α-aminonitrone-α-iminohydroxyloamine tautomerism of these products was studied theoretically and the structures of the synthesised compounds were confirmed by single crystal X-ray crystallographic analysis. The compounds were evaluated in vitro for their binding affinities to α(1) and α(2) adrenoceptors as well as imidazoline I(1) and I(2) receptors. The highest potencies at the α(2) adrenergic receptors were observed for compounds bearing biphenyl (4h, K(i) = 9 nM) and naphthyl (4i, K(i) = 92 nM) moieties. Compounds 4h and 4i were further tested in vivo for their cardiovascular and sedative-hypnotic effects in rats.

Synthesis and biological activities of 2-[(heteroaryl)methyl]imidazolines.

A series of 2-[(heteroaryl)methyl]imidazolines was synthesized and tested for their activities at α(1)- and α(2)-adrenoceptors and imidazoline I(1) and I(2) receptors. The most active 2-[(indazol-1-yl)methyl]imidazolines showed high or moderate affinities for α(1)- and α(2)-adrenoceptors. However, their intrinsic activities at α(2A)-adrenoceptors proved to be negligible. A selected 7-chloro derivative behaved as a potent α(1)-adrenoceptor antagonist and exhibited peripherally mediated hypotensive effects in rats.

Dexmedetomidine provides renoprotection against ischemia-reperfusion injury in mice.

INTRODUCTION: Acute kidney injury following surgery incurs significant mortality with no proven preventative therapy. We investigated whether the α2 adrenoceptor agonist dexmedetomidine (Dex) provides protection against ischemia-reperfusion induced kidney injury in vitro and in vivo. METHODS: In vitro, a stabilised cell line of human kidney proximal tubular cells (HK2) was exposed to culture medium deprived of oxygen and glucose. Dex decreased HK2 cell death in a dose-dependent manner, an effect attenuated by the α2 adrenoceptor antagonist atipamezole, and likely transduced by phosphatidylinositol 3-kinase (PI3K-Akt) signaling. In vivo C57BL/6J mice received Dex (25 µg/kg, intraperitoneal (i.p.)) 30 minutes before or after either bilateral renal pedicle clamping for 25 minutes or right renal pedicle clamping for 40 minutes and left nephrectomy. RESULTS: Pre- or post-treatment with Dex provided cytoprotection, improved tubular architecture and function following renal ischemia. Consistent with this cytoprotection, dexmedetomidine reduced plasma high-mobility group protein B1 (HMGB-1) elevation when given prior to or after kidney ischemia-reperfusion; pretreatment also decreased toll-like receptor 4 (TLR4) expression in tubular cells. Dex treatment provided long-term functional renoprotection, and even increased survival following nephrectomy. CONCLUSIONS: Our data suggest that Dex likely activates cell survival signal pAKT via α2 adrenoceptors to reduce cell death and HMGB1 release and subsequently inhibits TLR4 signaling to provide reno-protection.

Differential effects of interleukin-1beta and S100B on amyloid precursor protein in rat retinal neurons.

PURPOSE: Interleukin-1beta (IL-1beta) and S100B calcium binding protein B (S100B) have been implicated in the pathogenesis of Alzheimer's disease. Both are present in and around senile plaques and have been shown to increase levels of amyloid precursor protein (APP) mRNA in vitro. However, it is not known how either of these substances affects APP in vivo. METHODS: We have studied the effects of IL-1beta and S100B on the expression and processing of APP using a retinal-vitreal model. We have also investigated the effect of amyloid beta peptide (Abeta) on APP in the same system and the regulation of S100B production by Abeta and IL-1beta from retinal glial cells. RESULTS: Retinal ganglion cells constitutively express APP. However, after intravitreal injection of IL-1beta or Abeta there was a marked reduction in APP levels as detected by Western blotting and IL-1beta produced a decrease in APP immunoreactivity (IR). Nissl staining showed that the integrity of the injected retinas was unchanged after injection. Two days after S100B injection, there was a small reduction in APP-IR but this was accompanied by the appearance of some intensely stained large ganglion cells and there was some up-regulation in APP holoprotein levels on Western blot. Seven days post-S100B injection, these large, highly stained cells had increased in number throughout the retina. Injection of Abeta and IL-1beta also caused an increase in S100B production within the retinal Müller glial cells. CONCLUSION: These results support the hypothesis that S100B (a glial-derived neurotrophic factor) and IL-1beta (a pro-inflammatory cytokine) can modulate the expression and processing of APP in vivo and so may contribute to the progression of Alzheimer's disease.

Acute and chronic effects of intravitreally injected beta-amyloid on the neurotransmitter system in the retina.

The potential cytotoxic effect of aggregated Abeta(1-42) to neurons that express classical neurotransmitters, including acetylcholine, gamma-amino butyric acid, catecholamines and serotonin was assessed. The cholinergic system has been the central focus of the therapeutic drug strategies in amyloid-depositing pathologies such as Alzheimer's disease. Aggregated Abeta(1-42) has a multisystem cytotoxic effect causing non-specific reduction in immunoreactivity, dysfunction, or loss of retinal nerve cells. The extent of this was investigated using immunocytochemistry, TUNEL staining for apoptosis, and measurement of cell density as well as retinal surface area. There was a differential acute and/or chronic effect of Abeta on choline acetyltransferase, gamma-aminobutyric acid and 5-tryptamine hydroxylase systems, observed with the increasing time course of 6h to 5 months, and a bilateral/systemic effect. In contrast, the overall pattern of catecholaminergic system, as revealed by tyrosine hydroxylase immunoreactivity of the retina, appears to have remained relatively unaffected by Abeta (however this may reflect neuronal loss due to reduction in the retinal surface). This is the first in vivo evidence in a CNS model to show that not only all major neurotransmitter systems are differentially affected by Abeta aggregates but the effect may vary from one transmitter system to another under the same experimental conditions in situ and in a dose- and time-dependent manner.

Peri-infarct depolarizations lead to loss of perfusion in ischaemic gyrencephalic cerebral cortex.

In the light of accumulating evidence for the occurrence of spontaneous cortical spreading depression and peri-infarct depolarizations in the human brain injured by trauma or aneurysmal subarachnoid haemorrhage, we used DC electrode recording and laser speckle imaging to study the relationship between depolarization events and perfusion in the ischaemic, gyrencephalic brain. In 14 adult male cats anaesthetized with chloralose, one cerebral hemisphere was exposed and the middle cerebral artery occluded. Surface cortical perfusion in core and penumbral territories was imaged semiquantitatively at intervals of 13 s for 4 h. Cortical surface DC potential was recorded. Time interval between changes in DC potential and in perfusion was examined, and this comparison was repeated using microelectrodes for DC potential in five similar experiments in a second laboratory. Mean pre-occlusion perfusion was 11707 +/- 4581 units (equivalent to CBF (cerebral blood flow) approximately 40.5 +/- SD 14.4 ml/100 g/min), and fell on occlusion to 5318 +/- 2916 (CBF approximately 17.1 +/- 8.3), 5291 +/- 3407 (CBF approximately 17.0 +/- 10.1), and 6711 +/- 3271 (CBF approximately 22.2 +/- 9.6), quickly recovering to 8704 +/- 4581 (CBF approximately 29.5 +/- 14.4), 9741 +/- 4499 (CBF approximately 33.3 +/- 14.1) and 10 314 +/- 3762 (CBF approximately 35.4 +/- 11.4) on the core, intermediate and outer penumbral gyri, respectively. Mean perfusion later fell secondarily on core and intermediate gyri but, overall, was preserved on the outer (upper level of perfusion) gyrus during the period of observation. Pattern and severity of transient changes in perfusion associated with depolarization events varied with gyral location; falls in perfusion were sometimes profound and irreversible, and followed rather than preceded depolarization. In this model of occlusive stroke, reductions in perfusion linked to peri-infarct depolarization events contribute to secondary deterioration in penumbral areas. The findings suggest that such events play a central rather than a subsidiary role in cerebral infarction in the gyrencephalic brain.

Alterations in presenilin 1 processing by amyloid-beta peptide in the rat retina.

Accumulating evidence indicates that mutations in the presenilin 1 (PS1) gene are responsible for most cases of familial Alzheimer's disease (AD). Although its biological functions are not yet fully understood, it appears that PS1 plays a role in the processing and trafficking of the amyloid precursor protein (APP). However, little is known about factors that are involved in regulating the metabolism of PS1 especially in relation to AD pathology. In this study, we have examined the effect of optic nerve crush, intravitreal injection of the inflammatory agent lipopolysaccharide (LPS) or injection of amyloid beta(1-42) (A beta(1-42)) on the expression and processing of PS1 in the rat retina. We found that 48 h after injection of A beta(1-42) there was a dramatic alteration in the banding pattern of PS1 on Western blots, as indicated by marked changes in the levels of expression of some of its C- and N-terminal fragments in retinal homogenates. These results suggest an A beta(1-42)-induced potentiation of a non-specific stress-related but inflammation-independent alteration of processing of PS1 in this in vivo model.