scholarly journals Selective lowering of synapsins induced by oligomeric α-synuclein exacerbates memory deficits

2017 ◽  
Vol 114 (23) ◽  
pp. E4648-E4657 ◽  
Author(s):  
Megan E. Larson ◽  
Susan J. Greimel ◽  
Fatou Amar ◽  
Michael LaCroix ◽  
Gabriel Boyle ◽  
...  
Keyword(s):  
Amyloid Β ◽  
Gene Promoter ◽  
Camp Response Element Binding ◽  
Size Exclusion ◽  
Human Brain Tissue ◽  
Camp Response Element ◽  
Abnormal Accumulation ◽  
Exclusion Chromatography

Mounting evidence indicates that soluble oligomeric forms of amyloid proteins linked to neurodegenerative disorders, such as amyloid-β (Aβ), tau, or α-synuclein (αSyn) might be the major deleterious species for neuronal function in these diseases. Here, we found an abnormal accumulation of oligomeric αSyn species in AD brains by custom ELISA, size-exclusion chromatography, and nondenaturing/denaturing immunoblotting techniques. Importantly, the abundance of αSyn oligomers in human brain tissue correlated with cognitive impairment and reductions in synapsin expression. By overexpressing WT human αSyn in an AD mouse model, we artificially enhanced αSyn oligomerization. These bigenic mice displayed exacerbated Aβ-induced cognitive deficits and a selective decrease in synapsins. Following isolation of various soluble αSyn assemblies from transgenic mice, we found that in vitro delivery of exogenous oligomeric αSyn but not monomeric αSyn was causing a lowering in synapsin-I/II protein abundance. For a particular αSyn oligomer, these changes were either dependent or independent on endogenous αSyn expression. Finally, at a molecular level, the expression of synapsin genes SYN1 and SYN2 was down-regulated in vivo and in vitro by αSyn oligomers, which decreased two transcription factors, cAMP response element binding and Nurr1, controlling synapsin gene promoter activity. Overall, our results demonstrate that endogenous αSyn oligomers can impair memory by selectively lowering synapsin expression.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

scholarly journals Transcriptional Regulation of ZNF638 in Thermogenic Cells by the cAMP Response Element Binding Protein in Male Mice

2019 ◽  
Vol 3 (12) ◽  
pp. 2326-2340 ◽  
Author(s):  
Luce Perie ◽  
Narendra Verma ◽  
Lingyan Xu ◽  
Xinran Ma ◽  
Elisabetta Mueller
Keyword(s):  
Zinc Finger ◽  
Binding Protein ◽  
Regulatory Region ◽  
Camp Response Element Binding ◽  
Camp Response Element ◽  
Response Element ◽  
Element Binding Protein ◽  
Thermogenic Adipocytes

Abstract Zinc finger factors are implicated in a variety of cellular processes, including adipose tissue differentiation and thermogenesis. We have previously demonstrated that zinc finger protein 638 (ZNF638) is a transcriptional coactivator acting as an early regulator of adipogenesis in vitro. In this study, we show, to our knowledge for the first time, that, in vivo, ZNF638 abounds selectively in mature brown and subcutaneous fat tissues and in fully differentiated thermogenic adipocytes. Furthermore, gene expression studies revealed that ZNF638 is upregulated by cAMP modulators in vitro and by cold exposure and by pharmacological stimulation of β-adrenergic signaling in vivo. In silico analysis of the upstream regulatory region of the ZNF638 gene identified two putative cAMP response elements within 500 bp of the ZNF638 transcription start site. Detailed molecular analysis involving EMSA and chromatin immunoprecipitation assays demonstrated that cAMP response element binding protein (CREB) binds to these cAMP response element regions of the ZNF638 promoter, and functional studies revealed that CREB is necessary and sufficient to regulate the levels of ZNF638 transcripts. Taken together, these results demonstrate that ZNF638 is selectively expressed in mature thermogenic adipocytes and tissues and that its induction in response to classic stimuli that promote heat generation is mediated via CREB signaling, pointing to a possible novel role of ZNF638 in brown and beige fat tissues.

Differences in the association of insulin-like growth factor-I (IGF-I) and IGF-I variants with rat, sheep, pig, human and chicken plasma-binding proteins

1994 ◽  
Vol 140 (3) ◽  
pp. 475-482 ◽  
Author(s):  
A P D Lord ◽  
S E P Bastian ◽  
L C Read ◽  
P E Walton ◽  
F J Ballard
Keyword(s):  
Binding Proteins ◽  
Rat Plasma ◽  
Size Exclusion ◽  
Free Form ◽  
Igf Binding Proteins ◽  
Igf I ◽  
Exclusion Chromatography ◽  
Igf Binding

Abstract Associations between labelled insulin-like growth factors (IGFs) and IGF-binding proteins in plasma have been compared in the rat, sheep, human, pig and chicken. The IGFs tested were recombinant human IGF-I, the truncated variant, des(1–3)IGF-I, and LR3IGF-I, an extended form that had been engineered so as to minimize interactions with IGF-binding proteins. Marked species differences were demonstrated, notably that the IGF-I variants which exhibited extremely weak binding in rat plasma bound significantly in plasma from the other species. This result was shown both by size-exclusion chromatography of labelled IGFs added to plasma, in which the extent of variant IGF-I binding decreased in the order sheep>human>pig=chicken>rat, and by competition for labelled IGF-I binding in vitro, in which the order was pig=chicken>sheep>human>rat. Notwithstanding these differences, the two IGF-I variants showed only slight between-species binding differences when tested with purified rat, sheep and human IGF-binding protein-3. Ligand blotting experiments with plasma from the five species similarly showed a consistent pattern in that IGF-I binding was much greater than des(1–3)IGF-I binding, which in turn was greater than LR3 IGF-I binding. These experiments suggest first that IGF-binding properties measured after the removal of endogenous IGFs do not always reflect the situation with untreated plasma or in vivo, and secondly, the increased potencies of des(1–3)IGF-I and LR3 IGF-I in rat growth studies that have been ascribed to higher concentrations of these peptides in the free form cannot necessarily be extended to other species. Journal of Endocrinology (1994) 140, 475–482

scholarly journals Melanogenesis: A Search for Pheomelanin and Also, What Is Lurking Behind Those Dark Colors?

2019 ◽  
Author(s):  
Koen Vercruysse ◽  
Venise Govan
Keyword(s):  
Size Exclusion ◽  
Black Pigment ◽  
Main Reaction ◽  
Main Reaction Product ◽  
Light Yellow ◽  
Exclusion Chromatography ◽  
Co Precipitation ◽  
Mediated Oxidation

<p>We investigated the synthesis of melanin-like materials from DOPA, dopamine, norepinephrine and epinephrine in the presence of L-cysteine. We observed that L-cysteine delayed the formation of pigment from these catecholamines and that the presence of L-cysteine yielded darker-colored reaction mixtures. No reddish pigment was observed that would indicate the synthesis of pheomelanin-like material. The reactions were performed in the presence of Na<sub>2</sub>CO<sub>3</sub> and through the addition of CaCl<sub>2</sub> at the end of the reaction; the black, eumelanin-like material was co-precipitated with CaCO<sub>3</sub>. The remaining supernatant solutions were observed to be light-yellow to rusty-orange in color depending on the catecholamine used in the reaction. Size exclusion chromatography (SEC) analyses indicated that the removal of the black pigment left behind an oligomeric material that exhibited a strong absorbance band around 280nm. Our experimental and analytical observations prompt us to raise a number of points of discussion or hypotheses. 1) The presence of L-cysteine during the air-mediated oxidation of catecholamines leads to darker-colored pigments; not reddish or lighter-colored pigments that would visually resemble pheomelanin-like pigments, 2) SEC analyses suggested that the black pigment generated during the air-mediated oxidation of catecholamines is not necessarily the main reaction product, 3) The pre-formed, dark-colored pigments obtained through the air-mediated oxidative melanogenesis process can readily be deposited on insoluble mineral surfaces using an <i>in situ</i> co-precipitation procedure, 4) The air-mediated oxidation of catecholamines leads to a binary product that contains an insoluble, melanin-like substance and a soluble, oligo- or polymeric substance containing unoxidized precursor units, 5) The melanogenesis process leads to a binary product involving a non-covalently bonded combination of dark-colored pigment and a lighter-colored or colorless substance; the latter being understudied or ignored in the <i>in vitro</i> or <i>in vivo</i> studies of the melanogenesis process, 6) The kinetics of the melanogenesis process may determine the balance between insoluble and soluble components of the binary product generated; the slower the reaction the more dark-colored, insoluble pigment generated, 7) One should consider the possibility of intermolecularly, N-to-C, bonded units of catecholamines when evaluating the structure of melanins, polydopamines, etc. and 8) There is a need for a systematic study of the effect of amino acids (beyond just L-cysteine) and amines in general on the melanogenesis process.</p>

scholarly journals Exosomal tau with seeding activity is released from Alzheimer’s disease synapses, and seeding potential is associated with amyloid beta

2021 ◽  
Author(s):  
Emily Miyoshi ◽  
Tina Bilousova ◽  
Mikhail Melnik ◽  
Danyl Fakhrutdinov ◽  
Wayne W. Poon ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Human Pathology ◽  
Exclusion Chromatography ◽  
Flow Through ◽  
Aggregation Size ◽  
Synaptic Vesicle Release ◽  
Induced Aggregation

AbstractSynaptic transfer of tau has long been hypothesized from the human pathology pattern and has been demonstrated in vitro and in vivo, but the precise mechanisms remain unclear. Extracellular vesicles such as exosomes have been suggested as a mechanism, but not all tau is exosomal. The present experiments use a novel flow cytometry assay to quantify depolarization of synaptosomes by KCl after loading with FM2–10, which induces a fluorescence reduction associated with synaptic vesicle release; the degree of reduction in cryopreserved human samples equaled that seen in fresh mouse synaptosomes. Depolarization induced the release of vesicles in the size range of exosomes, along with tetraspanin markers of extracellular vesicles. A number of tau peptides were released, including tau oligomers; released tau was primarily unphosphorylated and C-terminal truncated, with Aβ release just above background. When exosomes were immunopurified from release supernatants, a prominent tau band showed a dark smeared appearance of SDS-stable oligomers along with the exosomal marker syntenin-1, and these exosomes induced aggregation in the HEK tau biosensor assay. However, the flow-through did not seed aggregation. Size exclusion chromatography of purified released exosomes shows faint signals from tau in the same fractions that show a CD63 band, an exosomal size signal, and seeding activity. Crude synaptosomes from control, tauopathy, and AD cases demonstrated lower seeding in tauopathy compared to AD that is correlated with the measured Aβ42 level. These results show that AD synapses release exosomal tau that is C-terminal-truncated, oligomeric, and with seeding activity that is enhanced by Aβ. Taken together with previous findings, these results are consistent with a direct prion-like heterotypic seeding of tau by Aβ within synaptic terminals, with subsequent loading of aggregated tau onto exosomes that are released and competent for tau seeding activity.

scholarly journals Preconditioning mice with activators of AMPK ameliorates ischemic acute kidney injury in vivo

2016 ◽  
Vol 311 (4) ◽  
pp. F731-F739 ◽  
Author(s):  
Wilfred Lieberthal ◽  
Meiyi Tang ◽  
Mark Lusco ◽  
Mersema Abate ◽  
Jerrold S. Levine
Keyword(s):  
Metabolic Stress ◽  
Kidney Injury ◽  
Camp Response Element Binding ◽  
Hexokinase Ii ◽  
Camp Response Element ◽  
Element Binding Protein ◽  
Proximal Tubular ◽  
Amp Activated Protein Kinase

This study had two objectives: 1) to determine whether preconditioning cultured proximal tubular cells (PTCs) with pharmacological activators of AMP-activated protein kinase (AMPK) protects these cells from apoptosis induced by metabolic stress in vitro and 2) to assess the effects of preconditioning mice with these agents on the severity of ischemic acute renal kidney injury (AKI) in vivo. We demonstrate that preconditioning PTCs with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or A-769662 reduces apoptosis of PTCs induced by subsequent stress. We also show that the reduction in cell death during metabolic stress associated with pretreatment by AMPK activators is associated with an increase in the cytosolic level of ATP, which is mediated by an increase in the rate of glycolysis. In addition, we provide evidence that the effect of AMPK activators on glycolysis is mediated, at least in part, by an increased uptake of glucose, and by the induction of hexokinase II (HK II) expression. Our data also show that the increased in HK II expression associated with preconditioning with AMPK activators is mediated by the activation (phosphorylation) of the cAMP-response element binding protein (CREB). We also provide entirely novel evidence that that A-79662 is substantially more effective than AICAR in mediating these alterations in PTCs in vitro. Finally, we demonstrate that preconditioning mice with AICAR or A-769662 substantially reduces the severity of renal dysfunction and tubular injury in a model of ischemic AKI in vivo and that the efficacy of AICAR and A-768662 in ameliorating ischemic AKI in vivo is comparable.

scholarly journals Extension of human GCSF serum half-life by the fusion of albumin binding domain

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Fatemeh Yadavar Nikravesh ◽  
Samira Shirkhani ◽  
Elham Bayat ◽  
Yeganeh Talebkhan ◽  
Esmat Mirabzadeh ◽  
...  
Keyword(s):  
Half Life ◽  
Animal Studies ◽  
Cytoplasmic Inclusion ◽  
Binding Domain ◽  
Size Exclusion ◽  
Albumin Binding ◽  
Exclusion Chromatography ◽  
Aggregation Formation

AbstractGranulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

scholarly journals A C-Terminally Truncated Variant of Neurospora crassa VDAC Assembles Into a Partially Functional Form in the Mitochondrial Outer Membrane and Forms Multimers in vitro

2021 ◽  
Vol 12 ◽  
Author(s):  
Fraser G. Ferens ◽  
William A. T. Summers ◽  
Ameet Bharaj ◽  
Jörg Stetefeld ◽  
Deborah A. Court
Keyword(s):  
Neurospora Crassa ◽  
Outer Membrane ◽  
Analytical Ultracentrifugation ◽  
Size Exclusion ◽  
Mitochondrial Outer Membrane ◽  
Voltage Dependent ◽  
Exclusion Chromatography ◽  
Selective Channel

The voltage-dependent anion-selective channel (VDAC) is a porin in the mitochondrial outer membrane (MOM). Unlike bacterial porins, several mitochondrial β-barrels comprise an odd number of β-strands, as is the case for the 19-β-stranded VDAC. Previously, a variant of a VDAC from Neurospora crassa, VDAC-ΔC, lacking the predicted 19th β-strand, was found to form gated, anion-selective channels in artificial membranes. In vivo, the two C-terminal β-strands (β18 and β19) in VDAC form a β-hairpin necessary for import from the cytoplasm into mitochondria and the β-signal required for assembly in the mitochondrial outer membrane resides in β19. The current study demonstrated that the putative 18-stranded β-barrel formed by VDAC-ΔC can be imported and assembled in the MOM in vivo and can also partially rescue the phenotype associated with the deletion of VDAC from a strain of N. crassa. Furthermore, when expressed and purified from Escherichia coli, VDAC-ΔC can be folded into a β-strand-rich form in decyl-maltoside. Size exclusion chromatography (SEC) alone or combined with multi-angle light scattering (SEC-MALS) and analytical ultracentrifugation revealed that, unlike full-length VDACs, VDAC-ΔC can self-organize into dimers and higher order oligomers in the absence of sterol.

scholarly journals Melanogenesis: A Search for Pheomelanin and Also, What Is Lurking Behind Those Dark Colors?

2019 ◽  
Author(s):  
Koen Vercruysse ◽  
Venise Govan
Keyword(s):  
Size Exclusion ◽  
Black Pigment ◽  
Main Reaction ◽  
Main Reaction Product ◽  
Light Yellow ◽  
Exclusion Chromatography ◽  
Co Precipitation ◽  
Mediated Oxidation

<p>We investigated the synthesis of melanin-like materials from DOPA, dopamine, norepinephrine and epinephrine in the presence of L-cysteine. We observed that L-cysteine delayed the formation of pigment from these catecholamines and that the presence of L-cysteine yielded darker-colored reaction mixtures. No reddish pigment was observed that would indicate the synthesis of pheomelanin-like material. The reactions were performed in the presence of Na<sub>2</sub>CO<sub>3</sub> and through the addition of CaCl<sub>2</sub> at the end of the reaction; the black, eumelanin-like material was co-precipitated with CaCO<sub>3</sub>. The remaining supernatant solutions were observed to be light-yellow to rusty-orange in color depending on the catecholamine used in the reaction. Size exclusion chromatography (SEC) analyses indicated that the removal of the black pigment left behind an oligomeric material that exhibited a strong absorbance band around 280nm. Our experimental and analytical observations prompt us to raise a number of points of discussion or hypotheses. 1) The presence of L-cysteine during the air-mediated oxidation of catecholamines leads to darker-colored pigments; not reddish or lighter-colored pigments that would visually resemble pheomelanin-like pigments, 2) SEC analyses suggested that the black pigment generated during the air-mediated oxidation of catecholamines is not necessarily the main reaction product, 3) The pre-formed, dark-colored pigments obtained through the air-mediated oxidative melanogenesis process can readily be deposited on insoluble mineral surfaces using an <i>in situ</i> co-precipitation procedure, 4) The air-mediated oxidation of catecholamines leads to a binary product that contains an insoluble, melanin-like substance and a soluble, oligo- or polymeric substance containing unoxidized precursor units, 5) The melanogenesis process leads to a binary product involving a non-covalently bonded combination of dark-colored pigment and a lighter-colored or colorless substance; the latter being understudied or ignored in the <i>in vitro</i> or <i>in vivo</i> studies of the melanogenesis process, 6) The kinetics of the melanogenesis process may determine the balance between insoluble and soluble components of the binary product generated; the slower the reaction the more dark-colored, insoluble pigment generated, 7) One should consider the possibility of intermolecularly, N-to-C, bonded units of catecholamines when evaluating the structure of melanins, polydopamines, etc. and 8) There is a need for a systematic study of the effect of amino acids (beyond just L-cysteine) and amines in general on the melanogenesis process.</p>

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