Soluble Neuregulin-1 from Microglia Enhances Amyloid Beta-induced Neuronal Death.

Neuregulin-1 (NRG-1) is a ligand of the epidermal growth factor receptor (erbB), and its interaction involves activation of the glutamatergic N-methyl-Daspartate receptor, which increases the expression of the ß2 subunit of the γ- aminobutyric acid receptor and subunits of the nicotinic acetylcholine receptor. In the dentate gyrus of 14-month-old Tg2576 mice, NRG-1 was strongly expressed compared with age-matched controls. The supernatant of oligomeric amyloid ß peptide (Aß42)-treated glial cells enhanced the Aß42;-induced cytotoxic effects, but the expression of Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand in microglial cells was not changed upon cytotoxic treatment. This suggests that the oligomeric form of Aß42 toxicity is not related to apoptosis, which is mediated by cell-to-cell interaction. During the 24-h incubation, the secretion of the soluble form of NRG-1 was increased, but interleukin 6 secretion was not changed. Further, soluble NRG-1 increased Aß42-induced toxicity. In conclusion, soluble NRG-1 significantly enhanced oligomeric Aß42-induced toxicity through the activation of endoplasmic reticulum stress by the increase of a phospho-translation initiation factor 2 alpha (p-eIF2α).

Facile "stop codon" method reveals elevated neuronal toxicity by discrete S87p-α-synuclein oligomers.

Herein, a new method for preparing phosphorylated proteins at specific sites has been applied to α-synuclein (α-Syn). Three different α-Syn species phosphorylated at Serine 87 (S87p-α-Syn), Serine 129 (S129p-α-Syn) and Serine 87/129 (S87p,129p-α-Syn) were prepared through the 'stop codon' method and verified by LC/MS/MS and immunoblotting. Each type of phosphorylated α-Syn was tested for oligomerization trends and cellular toxicity with dopamine (DA), Cu(2+) ions and pyridoxal 5'-phosphate. Aggregation trends induced by DA or DA/Cu(2+) were similar between phosphorylated and non-phosphorylated α-Syn in SDS-PAGE. However, except for the monomer, phosphorylated oligomers showed higher toxicity than the non-phosphorylated α-Syn (Np-α-Syn) oligomers via WST-1 assays when tested on SH-SY5Y human neuroblastoma cells. In particular, S87p-α-Syn and S87p,129p-α-Syn oligomers induced by DA/Cu(2+), showed higher toxicity than did S129p-α-Syn. When α-Syn was treated with pyridoxal 5'-phosphate in the presence of DA or Cu(2+) to determine aggregation effects, high inhibition effects were shown in both non-phosphorylated and phosphorylated versions. α-Syn co-incubated with DA or DA/Cu(2+) showed less cellular toxicity upon pyridoxal 5'-phosphate treatment, especially in the case of DA-induced Np-α-syn. This study supports that phosphorylated oligomers of α-Syn at residue 87 can contribute to neuronal toxicity and the pyridoxal 5'-phosphate can be used as an inhibitor for α-Syn aggregation.

Dopamine and Cu+/2+ can induce oligomerization of α-synuclein in the absence of oxygen: Two types of oligomerization mechanisms for α-synuclein and related cell toxicity studies.

α-Synuclein oligomers can induce neurotoxicity and are implicated in Parkinson's disease etiology and disease progression. Many studies have reported α-synuclein oligomerization by dopamine (DA) and transition metal ions, but few studies provide insight into joint influences of DA and Cu2+ . In this study, DA and Cu2+ were coadministered aerobically to measure α-synuclein oligomerization under these conditions. In the presence of oxygen, DA induced α-synuclein oligomerization in a dose-dependent manner. Cu+/2+ did not effect oligomerization in such a manner in the presence of DA. By electrophoresis, Cu2+ was found easily to induce oligomerization with DA. This implies that oligomerization invoked by DA is reversible in the presence of Cu2+, which appears to be mediated by noncovalent bond interactions. In the absence of oxygen, DA induced less oligomerization of α-synuclein, whereas DA/Cu2+ induced aerobic-level amounts of oligomers, suggesting that DA/Cu2+ induces oligomerization independent of oxygen concentration. Radical species were detected through electron paramagnetic resonance (EPR) spectroscopic analysis arising from coincubation of DA/Cu2+ with α-synuclein. Redox reactions induced by DA/Cu2+ were observed in multimer regions of α-synuclein oligomers through NBT assay. Cellular toxicity results confirm that, for normal and hypoxic conditions, copper or DA/Cu2+ can induce cell death, which may arise from copper redox chemistry. From these results, we propose that DA and DA/Cu2+ induce different mechanisms of α-synuclein oligomerization, cross-linking with noncovalent (or reversible covalent) bonding vs. likely radical-mediated covalent modification.

p35 deficiency accelerates HMGB-1-mediated neuronal death in the early stages of an Alzheimer's disease mouse model.

The activities of CDK5 and p35 are thought to be important in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). We studied the effect of p35 deletion in Tg2576 mice, which is an AD animal model. To obtain the desired mice, we crossed p35(-/-) with Tg2576 mice. The resulting p35(-/-)/Tg2576 (KO/Tg) mice displayed higher mortality rates and exhibited impaired spatial learning and memory at 6 months of age. Using immunohistochemical and biochemical approaches, we observed a reduction in the expression of pre- and post-synaptic markers such as NMDAR1, synaptophysin and GluR1. In addition, the intensity of MAP-2-positive dendrites extending from neuronal cell bodies was significantly decreased in KO/Tg mice compared with KO/WT and WT/Tg mice. We also detected increased neuronal cell death in the hippocampus, along with thinned and collapsed morphological changes in the alveus region and a dramatic increase in the number of microglial cells. Microglial infiltration in the hippocampus could result in the increased secretion of the soluble high mobility group box-1 protein (HMGB-1). The secretion of HMGB-1 is increased by Aß, and secretion of HMGB-1 promotes neuronal cell death. Moreover, we found that HMGB-1 secretion induced by Aß in KO/Tg mice gave rise to ER-mediated cell death. In summary, during the stages of KO/Tg mice model, the microglial infiltration and secretion of soluble HMGB-1 were significantly increased in the hippocampus. These conditions promote neuronal death, synaptic destruction and behavioral deficits.

Extremely selective "turn-on" fluorescence detection of hypochlorite confirmed by proof-of-principle neurological studies via esterase action in living cells.

Highly specific and sensitive fluorescence detection of hypochlorite in nonbiotic pure water (rapid "turn-on", ~400 fold, λ(em) ~ 560 nm) as well as in living neuronal cell cultures (neutral pH) involves oxidation of a 2-sulfide-2-benzoic acid pendent group in a new meso-thienyl-BODIPY donor-acceptor probe.

A novel, selective, and extremely responsive thienyl-based dual fluorogenic probe for tandem superoxide and Hg2+ chemosensing.

Novel, high "turn-on" Hg(2+) and O(2)(-) fluorescence behaviour (∼25-fold) with probes bearing [S(thi)N(py)] and [S(thi)N(py)N(py)] binding receptors, joined by oxidizable sulphides, may involve S-bound transient ROS species; such optical O(2)(-) behaviour operates moderately in neuroblastoma.

Phosphorylation of α-synuclein is crucial in compensating for proteasomal dysfunction.

α-Synuclein can be degraded by both the ubiquitin-proteasomal system and the chaperone-lysosomal system. However, the switching mechanism between the two pathways is not clearly understood. In our study, we investigated the mutual association between the binding of α-synuclein to heat shock cognate 70 and the lysosomal translocation of α-synuclein. Tyrosine phosphorylation of Y136 on α-synuclein increased when it bound to heat shock protein 70. We also found that tyrosine phosphorylation of α-synuclein can be regulated by focal adhesion kinase pp125 and protein tyrosine phosphatase 1B. Furthermore, protein tyrosine phosphatase 1B inhibitor protected dopaminergic neurons against cell death and rescued rotarod performance in a Parkinson's disease animal model. This study provides evidence that the regulation of Y136 phosphorylation of α-synuclein can improve behavioral performance and protect against neuronal death by promoting the turnover of lysosomal degradation of α-synuclein. As a result, protein tyrosine phosphatase 1B inhibitor may be used as a potential therapeutic agent against Parkinson's disease.

Novel sulphur-rich BODIPY systems that enable stepwise fluorescent O-atom turn-on and H2O2 neuronal system probing.

Bis-arylsulfide BODIPY systems were prepared and studied for multiple O-atom sensing (at 522 nm); 2- and 3-atom loading was optimal (50-fold, "turn on"). Neuronal studies showed greater H(2)O(2) sensitivity than 2',7'-dichlorofluorescein diacetate. The novel 1,3,6-trimethyl BODIPY formed as a biproduct under Lindsey conditions.

Interplay of salicylaldehyde, lysine, and M2+ ions on α-synuclein aggregation: cancellation of aggregation effects and determination of salicylaldehyde neurotoxicity.

In this study, α-synuclein was treated in vitro with salicylaldehyde (SA), lysine (lys) and M(n+) (Cu(2+) or Zn(2+)) in various ratios. SA induced aggregation of α-syn in the ratio of 1:500 (α-syn:SA) after incubation (pH 7.4, PBS buffer, 16-24h). Free lys can thus scavenge SA, inhibiting the aggregation of α-syn up to ∼63% (α-syn:SA:lys=1:1000:5000). When Cu(2+) and Zn(2+) are added to SA and α-syn, protein aggregation is induced. In the case of Zn(2+), the aggregation of α-syn increased to 74% (ratio=1:1000:50). Fluorescence studies support the production of protein-bound Zn(2+)-salicylaldimine species. For Cu(2+), aggregation of α-syn was shown (138%). Thus, possible protective or inducing effects of lys, Cu(2+) and Zn(2+) may exist with α-syn. α-Syn, SA and Cu(2+) can undergo complexation (fluorescence, CD and MALDI data). Cellular toxicity of SA (700µM), Zn(2+) (700µM) and Cu(2+) (700µM) on SH-SY5Y (1×10(5) cells) showed 9.8%, 38.0% and 14.4% compared to control values. Combinations showed more severe toxicities: 71.9% and 93.1% for SA (70µM)+Cu(2+) (700µM) and SA (70µM)+Zn(2+) (700µM), respectively, suggesting complexation itself may be toxic.

Amyloid precursor protein binding protein-1 modulates cell cycle progression in fetal neural stem cells.

Amyloid precursor protein binding protein-1 (APP-BP1) binds to the carboxyl terminus of the amyloid precursor protein (APP) and serves as the bipartite activation enzyme for the ubiquitin-like protein, NEDD8. In the present study, we explored the physiological role of APP-BP1 in the cell cycle progression of fetal neural stem cells. Our results show that cell cycle progression of the cells is arrested at the G1 phase by depletion of APP-BP1, which results in a marked decrease in the proliferation of the cells. This action of APP-BP1 is antagonistically regulated by the interaction with APP. Consistent with the evidence that APP-BP1 function is critical for cell cycle progression, the amount of APP-BP1 varies depending upon cell cycle phase, with culminating expression at S-phase. Furthermore, our FRET experiment revealed that phosphorylation of APP at threonine 668, known to occur during the G2/M phase, is required for the interaction between APP and APP-BP1. We also found a moderate ubiquitous level of APP-BP1 mRNA in developing embryonic and early postnatal brains; however, APP-BP1 expression is reduced by P12, and only low levels of APP-BP1 were found in the adult brain. In the cerebral cortex of E16 rats, substantial expression of both APP-BP1 and APP mRNAs was observed in the ventricular zone. Collectively, these results indicate that APP-BP1 plays an important role in the cell cycle progression of fetal neural stem cells, through the interaction with APP, which is fostered by phosphorylation of threonine 668.

Comparative proteomic analysis of virulent Korean Mycobacterium tuberculosis K-strain with other mycobacteria strain following infection of U-937 macrophage.

In Korea, the Mycobacterium tuberculosis K-strain is the most prevalent clinical isolates and belongs to the Beijing family. In this study, we conducted comparative porteomics of expressed proteins of clinical isolates of the K-strain with H37Rv, H37Ra as well as the vaccine strain of Mycobacterium bovis BCG following phagocytosis by the human monocytic cell line U-937. Proteins were analyzed by 2-D PAGE and MALDITOF-MS. Two proteins, Mb1363 (probable glycogen phosphorylase GlgP) and MT2656 (Haloalkane dehalogenase LinB) were most abundant after phagocytosis of M. tuberculosis K-strain. This approach provides a method to determine specific proteins that may have critical roles in tuberculosis pathogenesis.

Effects of the monomeric, oligomeric, and fibrillar Abeta42 peptides on the proliferation and differentiation of adult neural stem cells from subventricular zone.

The incidence of amyloid plaques, composed mainly of beta-amyloid peptides (Abeta), does not correlate well with the severity of neurodegeneration in patients with Alzheimer's disease (AD). The effects of Abeta(42) on neurons or neural stem cells (NSCs) in terms of the aggregated form remain controversial. We prepared three forms of oligomeric, fibrillar, and monomeric Abeta(42) peptides and investigated their effects on the proliferation and neural differentiation of adult NSCs, according to the degree of aggregation or concentration. A low micromolar concentration (1 micromol/L) of oligomeric Abeta(42) increased the proliferation of adult NSCs remarkably in a neurosphere assay. It also enhanced the neuronal differentiation of adult NSCs and their ability to migrate. These results provide us with valuable information regarding the effects of Abeta(42) on NSCs in the brains of patients with AD.