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 ~Beta conformation of polyglutamine track revealed by a crystal structure of Huntingtin N-terminal region with insertion of three histidine residues.

Huntington disease is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine (polyQ) expansion (> 35Q) in the first exon (EX1) of huntingtin protein (Htt). mHtt protein is thought to adopt one or more toxic conformation(s) that are involved in pathogenic interactions in cells . However, the structure of mHtt is not known. Here, we present a near atomic resolution structure of mHtt36Q-EX1. To facilitate crystallization, three histidine residues (3H) were introduced within the Htt36Q stretch resulting in the sequence of Q 7HQHQHQ 27. The Htt36Q3H region adopts α-helix, loop, β-hairpin conformations. Furthermore, we observed interactions between the backbone of the Htt36Q3H β-strand with the aromatic residues mimicking putative-toxic interactions with other proteins. Our findings support previous predictions that the expanded mHtt-polyQ region adopts a β-sheet structure. Detailed structural information about mHtt improves our understanding of the pathogenic mechanisms in HD and other polyQ expansion disorders and may form the basis for rational design of small molecules that target toxic conformations of disease-causing proteins.

Beta Amyloid ~Differential molecular chaperone response associated with various mouse adapted scrapie strains.

Prionoses are a group of neurodegenerative diseases characterized by misfolding of cellular prion protein (PrP(C)) and accumulation of its diseases specific conformer PrP(Sc) in the brain and neuropathologically, they can be associated with presence or absence of PrP amyloid deposits. Functional molecular chaperones (MCs) that constitute the unfolded protein response include heat shock proteins and glucose-regulated protein families. They protect intracellular milieu against various stress conditions including accumulation of misfolded proteins and oxidative stress, typical of neurodegenerative diseases. Little is known about the role of MCs in pathogenesis of prionoses in mammalian prion model systems. In this study we characterized MCs response pattern in mice infected with various mouse adapted scrapie strains. Rather than uniform upregulation of MCs, we encountered two distinctly different patterns of MCs response distinguishing ME7 and 87V strains from 22L and 139A strains. ME7 and 87V strains are known for the induction of amyloid deposition in infected animals, while in mice infected with 22L and 139A strains amyloid deposits are absent. MCs response pattern similar to that associated with amyloidogenic ME7 and 87V strains was also observed in APPPS1-21 Alzheimer's transgenic mice, which represent an aggressive model of cerebral amyloidosis caused by β-amyloid deposition. Our results highlight the probability that different mechanisms of MCs regulation exist driven by amyloidogenic and non-amyloidogenic nature of prion strains.

Beta Amyloid ~Brain regional correlation of amyloid-β with synapses and apolipoprotein E in non-demented individuals: potential mechanisms underlying regional vulnerability to amyloid-β accumulation.

To reveal the underlying mechanisms responsible for the regional vulnerability to amyloid-β (Aβ) accumulation prior to the development of Alzheimer's disease, we studied distribution of Aβ, apolipoprotein E (apoE), synaptic markers, and other molecules involved in Aβ metabolism in multiple brain areas of non-demented individuals. Twelve brain regions including neocortical, limbic, and subcortical areas were dissected from brains of non-demented individuals and extracted according to increasing insolubility by a sequential three-step method. The levels of Aβ40, Aβ42, apoE, APP, APP-CTFβ, BACE1, presenilin-1, neprilysin, insulysin, LRP1, LDLR, synaptophysin, PSD95, GFAP, and lactate were determined by ELISAs or enzymatic assays. The regional distribution of apoE showed moderate-to-strong inverse correlation with levels of Aβ, especially insoluble Aβ40. On the other hand, the regional distributions of synaptic markers, particularly PSD95, showed moderate-to-strong positive correlation with levels of Aβ, especially soluble Aβ40. The regional correlations between Aβ and LRP1, GFAP, or lactate were mild-to-moderate. Moderate-to-strong positive regional correlations were observed between apoE and GFAP or lactate and between PSD95 and LRP1. No significant regional correlations were detected between Aβ and APP, APP-CTFβ, BACE1, or presenilin-1, those involved in Aβ production. There were no significant negative regional correlations between Aβ and two major Aβ degrading enzymes, neprilysin and insulysin. These regional correlations remained consistent regardless of the degree of Aβ accumulation. The regional vulnerability to Aβ accumulation may be due to a net balance between two competing processes: (1) synapses involved in promoting the initial Aβ accumulation and (2) astrocyte-derived apoE involved in preventing Aβ accumulation.

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.

RESULTS:

EMSA probed with the PRE showed DNA-protein interaction in multiple nuclear extracts and in human brain tissue nuclear extract in a tissue-type specific manner. We identified transcription factors that are likely to bind the PRE, using competition gel shift and gel supershift: Activator protein 2 (AP2), nm23 nucleoside diphosphate kinase/metastatic inhibitory protein (PuF), and specificity protein 1 (SP1). These sites crossed a known single nucleotide polymorphism (SNP). EMSA with PRE mutants and promoter/reporter clone transfection analysis further implicated PuF in cells and extracts. Functional assays of mutant/reporter clone transfections were evaluated by ELISA of reporter protein levels. EMSA and ELISA results correlated by meta-analysis.

CONCLUSIONS:

We propose that PuF may regulate the APP gene promoter and that AD risk may be increased by interference with PuF regulation at the PRE. PuF is targeted by calcium/calmodulin-dependent protein kinase II inhibitor 1, which also interacts with the integrins. These proteins are connected to vital cellular and neurological functions. In addition, the transcription factor PuF is a known inhibitor of metastasis and regulates cell growth during development. Given that APP is a known cell adhesion protein and ferroxidase, this suggests biochemical links among cell signaling, the cell cycle, iron metabolism in cancer, and AD in the context of overall aging.

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.