Department of Neurology: Faculty Research and Presentations
http://hdl.handle.net/10675.2/860
2024-03-29T07:21:22ZMechanisms of Estrogen Neuroprotection in Stroke
http://hdl.handle.net/10675.2/317656
Mechanisms of Estrogen Neuroprotection in Stroke
Raz, Limor
17-β estradiol (17-β-E2) has been implicated to be neuroprotective, yet the mechanisms underlying 17-β-E2-mediated protection against stroke remain unclear. The purpose of the current study was to elucidate the role of 17-β-E2 in NADPH oxidase (NOX2) activation during ischemic reperfusion induction of superoxide (O2 -) in the hippocampus CA1 region following global cerebral ischemia (GCI) and to investigate the post-translational deacetylation of downstream pro-apoptotic factors by 17-β-E2. Using a 4-vessel occlusion model to induce GCI, we showed that neuronal NOX2 localizes to the membrane and that NADPH oxidase activity and O2 - production were rapidly and markedly attenuated by 17-β-E2 following reperfusion, in an estrogen receptor-dependent manner. Inhibition of NADPH oxidase activation via icv administration of a NOX2 competitive inhibitor, gp91ds-tat, strongly attenuated O2 - production and was neuroprotective. The increase of neuronal NOX2 and O2 - following cerebral ischemia was shown to require Rac1 activation, as administration of a Rac1 inhibitor (NSC23766) significantly attenuated these factors following stroke. Interestingly, we found that 17-β-E2 antioxidant ability to diminish neuronal NOX2-induced O2 - generation involves the attenuation of Rac1 activation. We also provide evidence for 17-β-E2 post-translational deacetylation of downstream pro-apoptotic p53 and a reduction of p53 transcriptional target, Puma. Our results revealed that p53 acetylation (activation) is markedly increased in ischemic animals 24h after reperfusion and that 17-β-E2 strongly attenuated that elevation, as well as total p53 protein levels. In support of this suggestion, we also found 17-β-E2 to strongly attenuate ischemia-mediated Puma upregulation, thus interfering with its transcription-dependent function. We further propose that 17-β-E2-induced attenuation of p53 levels may involve an upregulation in p53-Mdm2 interactions and p53 mediated degradation via the ubiquitination pathway. Lastly, we provide evidence showing that treatment with Gp91ds-tat, but not the scrambled tat peptide control, attenuated acetylation of downstream p53 and reduced levels of Puma, thus supporting O2 —p53 crosstalk signaling after stroke. Altogether, our studies reveal a novel, membrane-mediated antioxidant mechanism of 17-β-E2-induced neuroprotection via reduction of neuronal NOX2 activation and O2 - production, while providing evidence for 17-β-E2–mediated deacetylation and inactivation of p53, thereby protecting the hippocampus CA1 against cerebral ischemia.
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2011-04-01T00:00:00ZStructure-Functional Relationship Study of Glycosyltransferases
http://hdl.handle.net/10675.2/317266
Structure-Functional Relationship Study of Glycosyltransferases
Gu, Yihua
Gangliosides are a group of functional molecules and are synthesized by glycosyltransferases in a stepwise manner. Mechanism of ganglioside profile change in normal conditions or in diseases is not well understood. Gene regulations, protein structures and catalytic mechanisms of related glycosyltransferases may provide clues to elucidate this phenomenon. In our project, GD3-synthase is used as a model of glycosyltransferases for protein structure-functional relationship study. GD3-synthase is a key enzyme in the ganglioside biosynthetic network. It catalyzes the biosynthesis of ganglioside GD3, which is the entry substrate of biosynthesis of the b- and c- series gangliosides. GD3 is a minor ganglioside in adult vertebrate tissues, while it is highly expressed during embryonic development and in pathological conditions, such as cancer. GD3 is also involved in aging, cell proliferation, and cell differentiation. Aspects that are still poorly understood include: 1) the regulatory mechanisms for change of GD3 levels in normal condition and in diseases, 2) the structure of GD3-synthase, 3) how the structure is related to function, and 4) how the synthesis and degradation of the enzyme are regulated. To carry out the structure-functional relationship study on GD3-synthase, we developed a strategy to obtain a large amount of pure human GD3-synthase for crystallographic analysis. E. coli, yeast, and baculovirus systems were screened for selection of the expression system. Transmembrane domain-truncated human GD3-synthase was expressed in E. coli, but it aggregated into inclusion bodies. The refolded protein did not have enzyme activity. Human GD3-synthase could not be expressed in yeast cells, due to the presence of two yeast-specific stop codons. Although codon optimization was performed, the protein still could not be expressed in yeast cells. The soluble human GD3-synthase with enzymatic activity was expressed in Trichoplusia ni (T. ni) insect cells and secreted into the culture medium. The recombinant protein was purified from the culture medium with a yield of 1.45 mg/L. This is the first report of a procedure for expression and purification of GD3-synthase with a high yield, since the cDNA sequence was determined in 1994. As an alternative strategy, protein modeling was performed to study the structure-functional relationship of GD3-synthase. CstII, a bacterial sialyltransferase, was identified as a remote homologue of human GD3-synthase with a low sequence similarity. CstII and vertebrate sialyltransferases share a similar topology of protein structure, which makes it possible to build the structure of human GD3-synthase by using homology modeling. Sequence comparison (including primary sequence and secondary structure alignments) between CstII and human GD3-synthase was performed to identify the possible functional sites. Between sialylmotifs L and S, four highly conserved amino acid residues (Asn188, Pro189, Ser190, and Arg272) were identified. Protein sequence alignment of human sialyltransferases suggests that all conserved residues identified in our study are ST8Sia subfamily-specific. Functional analysis of these residues in human GD3-synthase was performed by using site-directed mutational analysis. Mutational analysis of these conserved residues suggests that Asn188, Ser190, and Arg272 are necessary for enzyme activity. The predicted 3-D structures of human GD3-synthase with docking of substrates support the data of mutational analysis and elucidate the contributions of these residues to the enzymatic activity, which suggests: 1) Asn188 is acceptor binding-related, 2) Ser190 functions to lock the acceptor substrate, and 3) Arg272 is acceptor binding-related. We also suggest that the protein modeling approach can be applied to structure-functional relationship studies only for those regions, which are highly conserved between vertebrate sialyltransferases and CstI/CstII, due to the low sequence similarity.
2008-05-01T00:00:00ZFocusing on Attention: The Effects of Working Memory Capacity and Load on Selective Attention
http://hdl.handle.net/10675.2/822
Focusing on Attention: The Effects of Working Memory Capacity and Load on Selective Attention
Ahmed, Lubna; de Fockert, Jan W.
Tsien, Joe Z.
Background: Working memory (WM) is imperative for effective selective attention. Distractibility is greater under conditions of high (vs. low) concurrent working memory load (WML), and in individuals with low (vs. high) working memory capacity (WMC). In the current experiments, we recorded the flanker task performance of individuals with high and low WMC during low and high WML, to investigate the combined effect of WML and WMC on selective attention.; Methodology/Principal Findings: In; Conclusions/Significance: The current findings show that limitations in WM resources, due to either WML or individual differences in WMC, affect the spatial distribution of attention. The difference in attentional constraining between high and low WMC individuals demonstrated in the current experiments helps characterise the nature of previously established associations between WMC and controlled attention.
2012-08-28T00:00:00ZSingle-Channel Electrophysiology Reveals a Distinct and Uniform Pore Complex Formed by α-Synuclein Oligomers in Lipid Membranes
http://hdl.handle.net/10675.2/818
Single-Channel Electrophysiology Reveals a Distinct and Uniform Pore Complex Formed by α-Synuclein Oligomers in Lipid Membranes
Schmidt, Felix; Levin, Johannes; Kamp, Frits; Kretzschmar, Hans; Giese, Armin; Botzel, Kai
Tsien, Joe Z.
Synucleinopathies such as Parkinson's disease, multiple system atrophy and dementia with Lewy bodies are characterized by deposition of aggregated α-synuclein. Recent findings indicate that pathological oligomers rather than fibrillar aggregates may represent the main toxic protein species. It has been shown that α-synuclein oligomers can increase the conductance of lipid bilayers and, in cell-culture, lead to calcium dyshomeostasis and cell death. In this study, employing a setup for single-channel electrophysiology, we found that addition of iron-induced α-synuclein oligomers resulted in quantized and stepwise increases in bilayer conductance indicating insertion of distinct transmembrane pores. These pores switched between open and closed states depending on clamped voltage revealing a single-pore conductance comparable to that of bacterial porins. Pore conductance was dependent on transmembrane potential and the available cation. The pores stably inserted into the bilayer and could not be removed by buffer exchange. Pore formation could be inhibited by co-incubation with the aggregation inhibitor baicalein. Our findings indicate that iron-induced α-synuclein oligomers can form a uniform and distinct pore species with characteristic electrophysiological properties. Pore formation could be a critical event in the pathogenesis of synucleinopathies and provide a novel structural target for disease-modifying therapy.
2012-08-03T00:00:00Z