Beta Amyloid ~IFN-γ Production by Amyloid β-Specific Th1 Cells Promotes Microglial Activation and Increases Plaque Burden in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the presence of amyloid-β (Aβ)-containing plaques, neurofibrillary tangles, and neuronal loss in the brain. Inflammatory changes, typified by activated microglia, particularly adjacent to Aβ plaques, are also a characteristic of the disease, but it is unclear whether these contribute to the pathogenesis of AD or are a consequence of the progressive neurodegenerative processes. Furthermore, the factors that drive the inflammation and neurodegeneration remain poorly understood. CNS-infiltrating T cells play a pivotal role in the pathogenesis of multiple sclerosis, but their role in the progression of AD is still unclear. In this study, we examined the role of Aβ-specific T cells on Aβ accumulation in transgenic mice that overexpress amyloid precursor protein and presenilin 1 (APP/PS1). We found significant infiltration of T cells in the brains of APP/PS1 mice, and a proportion of these cells secreted IFN-γ or IL-17. Aβ-specific CD4 T cells generated by immunization with Aβ and a TLR agonist and polarized in vitro to Th1-, Th2-, or IL-17-producing CD4(+) T cells, were adoptively transferred to APP/PS1 mice at 6 to 7 mo of age. Assessment of animals 5 wk later revealed that Th1 cells, but not Th2 or IL-17-producing CD4(+) T cells, increased microglial activation and Aβ deposition, and that these changes were associated with impaired cognitive function. The effects of Th1 cells were attenuated by treatment of the APP/PS1 mice with an anti-IFN-γ Ab. Our study suggests that release of IFN-γ from infiltrating Th1 cells significantly accelerates markers of diseases in an animal model of AD.

Beta Amyloid ~Dysregulation of Hypoxia-Inducible Factor by Presenilin/γ-Secretase Loss-of-Function Mutations.

Presenilin (PSEN) 1 and 2 are the catalytic components of the γ-secretase complex, which cleaves a variety of proteins, including the amyloid precursor protein (APP). Proteolysis of APP leads to the formation of the APP intracellular domain (AICD) and amyloid β that is crucially involved in the pathogenesis of Alzheimer's disease. Prolyl-4-hydroxylase-domain (PHD) proteins regulate the hypoxia-inducible factors (HIFs), the master regulators of the hypoxic response. We previously identified the FK506 binding protein 38 (FKBP38) as a negative regulator of PHD2. Genetic ablation of PSEN1/2 has been shown to increase FKBP38 protein levels. Therefore, we investigated the role of PSEN1/2 in the oxygen sensing pathway using a variety of genetically modified cell and mouse lines. Increased FKBP38 protein levels and decreased PHD2 protein levels were found in PSEN1/2-deficient mouse embryonic fibroblasts and in the cortex of forebrain-specific PSEN1/2 conditional double knock-out mice. Hypoxic HIF-1α protein accumulation and transcriptional activity were decreased, despite reduced PHD2 protein levels. Proteolytic γ-secretase function of PSEN1/2 was needed for proper HIF activation. Intriguingly, PSEN1/2 mutations identified in Alzheimer patients differentially affected the hypoxic response, involving the generation of AICD. Together, our results suggest a direct role for PSEN in the regulation of the oxygen sensing pathway via the APP/AICD cleavage cascade.

Beta Amyloid ~γ-Secretase Modulators: Can We Combine Potency with Safety?

γ-Secretase modulation has been proposed as a potential disease modifying anti-Alzheimer's approach. γ-Secretase modulators (GSMs) cause a product shift from the longer amyloid-beta (Aβ) peptide isoforms to shorter, more soluble, and less amyloidogenic isoforms, without inhibiting APP or Notch proteolytic processing. As such, modulating γ-secretase may avoid some of the adverse effects observed with γ-secretase inhibitors. Since the termination of the GSM tarenfurbil in 2008 due to negative phase III trial results, a considerable progress has been made towards more potent and better brain penetrable compounds. However, an analysis of their lipophilic efficiency indices indicates that their increased potency can be largely attributed to their increased lipophilicity. The need for early and chronic dosing with GSMs will require high-safety margins. This will be a challenge to achieve with the current, highly lipophilic GSMs. We will demonstrate that by focusing on the drug-like properties of GSMs, a combination of high in vitro potency and reduced lipophilicity can be achieved and does result in better tolerated compounds. The next hurdle will be to translate this knowledge into GSMs which are highly efficacious and safe in vivo.

Beta Amyloid ~PuF, an antimetastatic and developmental signaling protein, interacts with the Alzheimer's amyloid-beta precursor protein via a tissue-specific proximal regulatory element (PRE).

BACKGROUND: Alzheimer's disease (AD) is intimately tied to amyloid-beta (Abeta) peptide. Extraneuronal brain plaques consisting primarily of Abeta aggregates are a hallmark of AD. Intraneuronal Abeta subunits are strongly implicated in disease progression. Protein sequence mutations of the Abeta precursor protein (APP) account for a small proportion of AD cases, suggesting that regulation of the associated gene (APP) may play a more important role in AD etiology. The APP promoter possesses a novel 30 nucleotide sequence, or "proximal regulatory element" (PRE), at -76/-47, from the +1 transcription start site that confers cell type specificity. This PRE contains sequences that make it vulnerable to epigenetic modification and may present a viable target for drug studies. We examined PRE-nuclear protein interaction by gel electrophoretic mobility shift assay (EMSA) and PRE mutant EMSA. This was followed by functional studies of PRE mutant/reporter gene fusion clones.

Beta Amyloid~Encephalopathy: A Vicious Cascade following Forebrain Ischemia and Hypoxia.

Post ischemic/hypoxic encephalopathy is a progressive and widespread damage syndrome in human brain, which includes production of new ischemic foci as well as neurodegeneration associated with accumulation of amyloid protein (Aβ), which emerges within days after the primary ischemic or hypoxic ictus. Patients may suddenly suffer severe dementia and Parkinson's syndrome after a symptom-free period averaging 2 weeks following resuscitation. Death of neurons in the cortex, limbic system, globus pallidus (GP) and substantia nigra (SN) and damage to white matter are responsible. From experimental studies in animals evidence is obtained to reveal the mechanisms. Injured endothelia and activated platelets lead to secondary injury via thrombosis and vasoconstriction resulting in infarction and new foci of necrosis. Blood-brain barrier (BBB) breakdown allows penetration of blood-borne toxic substances into brain resulting in neuronal degeneration and enhanced inflammatory destruction. These secondary injuries happen within two weeks after moderate global ischemia. As these pathological changes cycle between the vascular and neuronal compartments, the damage expands and worsens. Aβ, β amyloid precursor protein (βAPP) and the inflammation mediator cyclooxygenase-2 (COX2) as well as γ-aminobutyric acid (GABA) system degeneration participate in producing secondary injury. Thus, implementing multi-targeted prophylaxis before or at the brain-at-risk stage is desirable. A combination of protecting endothelia, inhibiting platelet activity and improving cerebral circulation is a fundamental strategy to block this vicious cascade, thereby ameliorating or preventing the encephalopathy.

Beta Amyloid ~Size-controllable networked neurospheres as a 3D neuronal tissue model for Alzheimer's disease studies.

Intensive in vitro studies on the neurotoxicity of amyloid beta have been conducted for decades; however, a three-dimensional neuronal tissue model for Alzheimer's disease has not yet been achieved. In this study, we developed size-controllable networked neurospheres comprised of cerebral cortical neuronal cells that mimics the cytoarchitecture of the cortical region of the brain. The toxicity of amyloid beta on the neurosphere model was assessed quantitatively and qualitatively. Decreased cell viability after amyloid beta exposure was demonstrated using MTT and live/dead assays. Neurite degeneration after amyloid beta exposure was evident in both SEM and fluorescence images. Ultrastructural features of apoptotic neurons were analyzed and quantitative analysis of synapsin II concentration and an acetylcholine assay were also performed. The three-dimensional neurospheres, produced using a concave microwell array, are a potential in vitro model for Alzheimer's disease studies.

Beta Amyloid ~Hydrophobic Interaction Drives Surface-Assisted Epitaxial Assembly of Amyloid-like Peptides.

The molecular mechanism of epitaxial fibril formation has been investigated for GAV-9 (NH3+-VGGAVVAGV-CONH2), anamyloid-like peptide extracted from a consensus sequence of amyloidogenic proteins, which assembles with very different morphologies, "upright" on mica and "flat" on the highly-oriented pyrolytic graphite (HOPG). Our all-atom molecular dynamics simulations reveal that the strong electrostatic interaction induces the "upright" conformation on mica, whereas the hydrophobic interaction favors the "flat" conformation on HOPG. We also show that the epitaxial pattern on mica is ensured by the lattice matching between the anisotropic binding sites of the basal substrate and the molecular dimension of GAV-9, accompanied with a long-range order of well-defined beta-strands. Furthermore, the binding free energy surfaces indicate that the longitudinal assembly growth is predominantly driven by the hydrophobic interaction along the longer crystallographic unit cell direction of mica. These findings provide a molecular basis for the surface-assisted molecular assembly, which might also be useful for the design of de novo nanodevices.

Beta Amyloid ~Synthesis of the Alzheimer drug Posiphen into its primary metabolic products (+)-N1-norPosiphen, (+)-N8-norPosiphen and (+)-N1, N8-bisnorPosiphen, their inhibition of amyloid precursor protein, α-synuclein synthesis, interleukin-1β release, and cholinergic action.

A major pathological hallmark of Alzheimer disease (AD) is the appearance in the brain of senile plaques that are primarily composed of aggregated forms of β-amyloid peptide (Aβ) that derive from amyloid precursor protein (APP). Posiphen (1) tartrate is an experimental AD drug in current clinical trials that reduces Aβ levels by lowering the rate of APP synthesis without toxicity. To support the clinical development of Posiphen (1) and elucidate its efficacy, its three major metabolic products, (+)-N1-norPosiphen (15), (+)-N8-norPosiphen (17) and (+)-N1, N8-bisnorPosiphen (11), were required in high chemical and optical purity. The efficient transformation of Posiphen (1) into these metabolic products, 15, 17 and 11, is described. The biological activity of these metabolites together with Posiphen (1) and and its enantiomer, the AD drug candidate (-)-phenserine (2), was assessed against APP, α-synuclein and classical cholinergic targets. All the compounds potently inhibited the generation of APP and a-synuclein in neuronal cultures. In contrast, metabolites 11 and 15, and (-)-phenserine (2) but not Posiphen (1) or 17, possessed acetylcholinesterase inhibitory action and no compounds bound either nicotinic or muscarinic receptors. As Posiphen (1) lowered CSF markers of inflammation in a recent clinical trial, the actions of 1 and 2 on proinflammatory cytokine interleukin (IL)-1β release human peripheral bloodmononuclear cells was evaluated, and found to be potently inhibited by both agents.

Beta Amyloid ~Molecular Interaction study of N1-p-fluorobenzyl-cymserine with TNF-α, p38 kinase and JNK kinase.

Alzheimer's disease (AD) is an age-related neurodegenerative disease distinguished by progressive memory loss and cognitive decline. It is accompanied by classical neuropathological changes, including cerebral deposits of amyloid-beta peptide (Aβ)-containing senile plaques, neurofibrillary tangles (NFTs) of phosphorylated tau (p-tau), and clusters of activated glial cells. Postmortem studies strongly support a critical role for neuroinflammation in the pathogenesis of AD, with activated microglia and reactive astrocytes surrounding senile plaques and NFTs. These are accompanied by an elevated expression of inflammatory mediators that further drives Ab and p-tau generation. Although epidemiological and experimental studies suggested that long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may lessen AD risk by mitigating inflammatory responses, primary NSAID treatment trials of AD have not proved successful. Elevated systemic butyrylcholinesterase (BuChE) levels have been considered a marker of low-grade systemic inflammation, and BuChE levels are reported elevated in AD brain. Recent research indicates that selective brain inhibition of BuChE elevates acetylcholine (ACh) and augments cognition in rodents free of the characteristic undesirable actions of acetylcholinesterase-inhibitors (AChE-Is). Hence, centrally active BuChE-selective-inhibitors, cymserine analogs, have been developed to test the hypothesis that BuChE-Is would be efficacious and better tolerated than AChE-Is in AD. The focus of the current study was to undertake an in-silico evaluation of an agent to assess its potential to halt the self-propagating interaction between inflammation, Ab and p-tau generation. Molecular docking studies were performed between the novel BuChE-I, N1-p-fluorobenzyl-cymserine (FBC) and inflammatory targets to evaluate the potential of FBC as an inhibitor of p38, JNK kinases and TNF-a with respect to putative binding free energy and IC50 values. Our in-silico studies support the ability of FBC to bind these targets in a manner supportive of anti-inflammatory action that is subject to molecular dynamics and physiochemical studies for auxiliary confirmation.

Beta Amyloid ~In Vitro and Mechanistic Studies of an Anti-Amyloidogenic Self-Assembled Cyclic D,L-α-Peptide Architecture.

Misfolding of the Aβ protein and its subsequent aggregation into toxic oligomers are related to Alzheimer's disease. Although peptides of various sequences can self-assemble into amyloid structures, these structures share common three-dimensional features that may promote their cross-reaction. Given the significant similarities between amyloids and the architecture of self-assembled cyclic D,L-α-peptide, we hypothesized that the latter may bind and stabilize a non-toxic form of Aβ, so preventing its aggregation into toxic forms. By screening a focused library of six-residue cyclic D,L-α-peptides and optimizing the activity of a lead peptide, we found one cyclic D,L-α-peptide (CP-2) that interacts strongly with Aβ and inhibits its aggregation. In transmission electron microscopy and optimized thioflavin T assays, CP-2 inhibits the formation of Aβ aggregates, entirely disassembles pre-formed aggregated and fibrillar Aβ, and protects rat pheochromocytoma PC12 cells from Aβ toxicity, without inducing any toxicity by itself. Using various immunoassays, circular dichroism spectroscopy, photo-induced cross-linking of unmodified proteins (PICUP) combined with SDS/PAGE, and NMR, we probed the mechanisms underlying CP-2's anti-amyloidogenic activity. NMR spectroscopy indicates that CP-2 interacts with Aβ through its self-assembled conformation and induces weak secondary structure in Aβ. Upon co-incubation, CP-2 changes the aggregation pathway of Aβ and alters its oligomer distribution by stabilizing low-molecular-weight species. Our results support studies suggesting that toxic early oligomeric states of Aβ may be composed of antiparallel β-peptide structures and that the interaction of Aβ with CP-2 promotes formation of more benign parallel β-structures. Further studies will show whether these kinds of abiotic cyclic D,L-α-peptides are also beneficial as an intervention in related in vivo models.