Hyunjeong Liew

Effects of the monomeric, oligomeric, and fibrillar Aβ42 peptides on the proliferation and differentiation of adult neural stem cells from subventricular zone

The incidence of amyloid plaques, composed mainly of β‐amyloid peptides (Aβ), does not correlate well with the severity of neurodegeneration in patients with Alzheimer’s disease (AD). The effects of Aβ42 on neurons or neural stem cells (NSCs) in terms of the aggregated form remain controversial. We prepared three forms of oligomeric, fibrillar, and monomeric Aβ42 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 μmol/L) of oligomeric Aβ42 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 Aβ42 on NSCs in the brains of patients with AD.

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 phopshorylation of threonine 668.

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 Alzheimers 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 Aβ, and secretion of HMGB-1 promotes neuronal cell death. Moreover, we found that HMGB-1 secretion induced by Aβ 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.

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.

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 Parkinsons 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 Parkinsons disease.

Soluble Neuregulin-1 as a Diagnostic Biomarker for AlzheimerâÂÂs Disease

Diagnosis using a biomarker is a faster and cheaper than brain imaging. Diagnostic biomarkers are chosen based on the characteristics of the disease, specificity, sensitivity, and stability during all disease stages. For this reason, previous candidates with insoluble form in a pathophysiological stage are not useful as biomarkers for the early stage of a neurodegenerative disease. In this study, we explored the possibility of using soluble proteins in cerebrospinal fluid, blood, or other peripheral materials as diagnostic biomarkers, in particular, the availability of soluble neuregulin-1 in blood.

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 β2 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 β peptide (Aβ42)-treated glial cells enhanced the Aβ42;-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 Aβ42 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 Aβ42-induced toxicity. In conclusion, soluble NRG-1 significantly enhanced oligomeric Aβ42-induced toxicity through the activation of endoplasmic reticulum stress by the increase of a phospho-translation initiation factor 2 alpha (p-eIF2α).

P4-287: Tyr125 phosphorylation of α-synuclein is the key of turnover between proteasomal and lysosomal degradation pathway

disease. Particularly, neuron cells in brains of neurodegenerative disease contained autophagic vacuoles as well as precipitates (aggregates) of various proteins characteristically. We want to know what kind of mechanism for survival break out in autophagic condition or for blocking of neuron cell death, what mechanism is triggered. Methods: In autophagic condition, we definitely detected that alpha-synuclein from cytoplasm is translocated to nucleus in neuron cells. In this study, we describe for the first time physical, direct interaction of alpha-synuclein with nuclear cargo protein KPNA that is the nuclear translocation mechanism of alpha-synuclein. We explored the functional consequences of this interaction on neuron cell survival by cell based transfection and gene knock-down experiments(Sh RNA). It was confirmed using GST-pull down assay, Immunoprecipitation, FRET methods. Results: With confocal microscopy, we definitely detected a-synuclein can translocate to nucleus in autophagy. In the results of GST-pull down assay and Immunoprecipitation, a-synuclein can interact with KPNA6 and this interaction cannot detected to between recombinant proteins. We also observed this interaction by FRET (Fluorescence resonance energy transfer). Conclusions: For passing the wicket NPC (Nuclear Pore Complex) a-synuclein bind with KPNA 6. This interaction needs some modification because just recombinant a-synuclein cannot interact with KPNA 6. Nuclear translocation resulted to anti-proliferative effect and furthermore it was related to survival mechanism.