Synaptosomes: new vesicles for neuronal mitochondrial transplantation

Abstract Background Mitochondrial dysfunction is a critical factor in the onset and progression of neurodegenerative diseases. Recently, mitochondrial transplantation has been advised as an innovative and attractive strategy to transfer and replace damaged mitochondria. Here we propose, for the first time, to use rat brain extracted synaptosomes, a subcellular fraction of isolated synaptic terminal that contains mitochondria, as mitochondrial delivery systems. Results Synaptosome preparation was validated by the presence of Synaptophysin and PSD95. Synaptosomes were characterized in terms of dimension, zeta potential, polydispersity index and number of particles/ml. Nile Red or CTX-FITCH labeled synaptosomes were internalized in LAN5 recipient cells by a mechanism involving specific protein–protein interaction, as demonstrated by loss of fusion ability after trypsin treatment and using different cell lines. The loading and release ability of the synaptosomes was proved by the presence of curcumin both into synaptosomes and LAN5 cells. The vitality of mitochondria transferred by Synaptosomes was demonstrated by the presence of Opa1, Fis1 and TOM40 mitochondrial proteins and JC-1 measurements. Further, synaptosomes deliver vital mitochondria into the cytoplasm of neuronal cells as demonstrated by microscopic images, increase of TOM 40, cytochrome c, Hexokinase II mitochondrial proteins, and presence of rat mitochondrial DNA. Finally, by using synaptosomes as a vehicle, healthy mitochondria restored mitochondrial function in cells containing rotenone or CCCp damaged mitochondria. Conclusions Taken together these results suggest that synaptosomes can be a natural vehicle for the delivery of molecules and organelles to neuronal cells. Further, the replacement of affected mitochondria with healthy ones could be a potential therapy for treating neuronal mitochondrial dysfunction-related diseases.

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Synaptosomes: New Vesicles for Neuronal Mitochondrial Transplantation

10.21203/rs.3.rs-89848/v1 ◽

2020 ◽

Author(s):

Pasquale Picone ◽

Gaetana Porcelli ◽

Celeste Caruso Bavisotto ◽

Domenico Nuzzo ◽

Giacoma Galizzi ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Neuronal Cells ◽

Nile Red ◽

Critical Factor ◽

Specific Protein ◽

Mitochondrial Proteins ◽

Hexokinase Ii ◽

Protein Protein Interaction ◽

First Time ◽

Fusion Ability

Abstract Background: Mitochondrial dysfunction is a critical factor in the onset and progression of neurodegenerative diseases. Recently, mitochondrial transplantation has been advised as an innovative and attractive strategy to transfer and replace damaged mitochondria. Here we propose, for the first time, to use rat brain extracted synaptosomes, subcellular fraction of isolated synaptic terminal that contain mitochondria, as mitochondrial delivery systems. Results: Synaptosomes preparation was validated by the presence of Synaptophysin and PSD95. Syn aptosomes were characterized in terms of dimension, zeta potential, polydispersity index and number of particles/mL. Nile Red or CTX-FITCH labeled synaptosomes were internalized in LAN5 recipient cells by a mechanism involving specific protein-protein interaction, as demonstrated by loss of fusion ability after trypsin treatment and using different cell lines. The loading and release ability of the synaptosomes was proved by the presence of curcumin both into synaptosomes and LAN5 cells. The vitality of mitochondria transferred by Synaptosomes was demonstrated by the presence of Opa1, Fis1 and TOM40 mitochondrial proteins and JC-1 measurements. Further, synaptosomes deliver vital mitochondria into the cytoplasm of neuronal cells as demonstrated by microscopic images, increase of TOM 40, cytochrome c, Hexokinase II mitochondrial proteins, and presence of rat mitochondrial DNA. Finally, by using synaptosomes as vehicle, healthy mitochondria restored mitochondrial function in cells containing rotenone or CCCp damaged mitochondria. Conclusions: Taken together these results suggest that synaptosomes can be a natural vehicle for the delivery of molecules and organelles to neuronal cells. Further, replacement of affected mitochondria with healthy ones could be a potential therapy for the treatment of neuronal mitochondrial dysfunction-related diseases.

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Transcriptional landscape of cellular networks reveal interactions driving the dormancy mechanisms in cancer

Scientific Reports ◽

10.1038/s41598-021-94005-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Dilara Uzuner ◽

Yunus Akkoç ◽

Nesibe Peker ◽

Pınar Pir ◽

Devrim Gözüaçık ◽

...

Keyword(s):

Cancer Cells ◽

Regulatory Networks ◽

Metastatic Cancer ◽

Interaction Network ◽

Current Treatment ◽

Cellular Mechanisms ◽

Protein Protein Interaction ◽

Cancer Dormancy ◽

Protein Protein Interaction Networks ◽

First Time

AbstractPrimary cancer cells exert unique capacity to disseminate and nestle in distant organs. Once seeded in secondary sites, cancer cells may enter a dormant state, becoming resistant to current treatment approaches, and they remain silent until they reactivate and cause overt metastases. To illuminate the complex mechanisms of cancer dormancy, 10 transcriptomic datasets from the literature enabling 21 dormancy–cancer comparisons were mapped on protein–protein interaction networks and gene-regulatory networks to extract subnetworks that are enriched in significantly deregulated genes. The genes appearing in the subnetworks and significantly upregulated in dormancy with respect to proliferative state were scored and filtered across all comparisons, leading to a dormancy–interaction network for the first time in the literature, which includes 139 genes and 1974 interactions. The dormancy interaction network will contribute to the elucidation of cellular mechanisms orchestrating cancer dormancy, paving the way for improvements in the diagnosis and treatment of metastatic cancer.

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Genome-wide identification of the GATA transcription factor family and their expression patterns under temperature and salt stress in Aspergillus oryzae

AMB Express ◽

10.1186/s13568-021-01212-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Chunmiao Jiang ◽

Gongbo Lv ◽

Jinxin Ge ◽

Bin He ◽

Zhe Zhang ◽

...

Keyword(s):

Salt Stress ◽

Expression Patterns ◽

Interaction Network ◽

Protein Protein Interaction ◽

Genome Wide ◽

Gata Transcription Factor ◽

Starting Point ◽

Evolutionary Features ◽

First Time

AbstractGATA transcription factors (TFs) are involved in the regulation of growth processes and various environmental stresses. Although GATA TFs involved in abiotic stress in plants and some fungi have been analyzed, information regarding GATA TFs in Aspergillusoryzae is extremely poor. In this study, we identified and functionally characterized seven GATA proteins from A.oryzae 3.042 genome, including a novel AoSnf5 GATA TF with 20-residue between the Cys-X2-Cys motifs which was found in Aspergillus GATA TFs for the first time. Phylogenetic analysis indicated that these seven A. oryzae GATA TFs could be classified into six subgroups. Analysis of conserved motifs demonstrated that Aspergillus GATA TFs with similar motif compositions clustered in one subgroup, suggesting that they might possess similar genetic functions, further confirming the accuracy of the phylogenetic relationship. Furthermore, the expression patterns of seven A.oryzae GATA TFs under temperature and salt stresses indicated that A. oryzae GATA TFs were mainly responsive to high temperature and high salt stress. The protein–protein interaction network of A.oryzae GATA TFs revealed certain potentially interacting proteins. The comprehensive analysis of A. oryzae GATA TFs will be beneficial for understanding their biological function and evolutionary features and provide an important starting point to further understand the role of GATA TFs in the regulation of distinct environmental conditions in A.oryzae.

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Oleate prevents palmitate-induced mitochondrial dysfunction, insulin resistance and inflammatory signaling in neuronal cells

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/j.bbamcr.2014.04.004 ◽

2014 ◽

Vol 1843 (7) ◽

pp. 1402-1413 ◽

Cited By ~ 53

Author(s):

Bumsup Kwon ◽

Han-Kyu Lee ◽

Henry W. Querfurth

Keyword(s):

Insulin Resistance ◽

Mitochondrial Dysfunction ◽

Neuronal Cells ◽

Inflammatory Signaling

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Retinoschisis and Norrie Disease: A missing link

10.21203/rs.3.rs-322315/v1 ◽

2021 ◽

Author(s):

Rahini Rajendran ◽

Sudha Dhandayuthapani ◽

Subbulakshmi Chidambaram ◽

Hemavathy Nagarajan ◽

Umashankar Vetrivel ◽

...

Keyword(s):

Mass Spectrometry ◽

Functional Relationship ◽

Direct Interaction ◽

Specific Protein ◽

Knock Out ◽

Norrie Disease ◽

Protein Protein Interaction ◽

Protein Interactome ◽

Molecular Relationships

Abstract Objective: Retinoschisis and Norrie disease are X-linked recessive retinal disorders caused by mutations in RS1 and NDP genes respectively. Both are likely to be monogenic and no locus heterogeneity has been reported. However, there are reports showing overlapping features of Norrie disease and retinoschisis in a NDP knock-out mouse model and also the involvement of both the genes in retinoschisis patients. Yet, the exact molecular relationships between the two disorders have still not been understood. The study investigated the association between retinoschisin (RS1) and norrin (NDP) using in vitro and in silico approaches. Specific protein-protein interaction between RS1 and NDP was analyzed in human retina by co-immunoprecipitation assay and MALDI-TOF mass spectrometry. STRING database was used to explore the functional relationship. Result: Co-immunoprecipitation demonstrated lack of a direct interaction between RS1 and NDP and was further substantiated by mass spectrometry. However, STRING revealed a potential indirect functional association between the two proteins. Progressively, our analyses indicate that FZD4 protein interactome via PLIN2 as well as the MAP kinase signaling pathway to be a likely link bridging the functional relationship between retinoschisis and Norrie disease.

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An Application of Fit Quality to Screen MDM2/p53 Protein-Protein Interaction Inhibitors

Molecules ◽

10.3390/molecules23123174 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3174 ◽

Cited By ~ 1

Author(s):

Xin Xue ◽

Gang Bao ◽

Hai-Qing Zhang ◽

Ning-Yi Zhao ◽

Yuan Sun ◽

...

Keyword(s):

Protein Interaction ◽

Drug Target ◽

P53 Protein ◽

Pharmacophore Model ◽

Cellular Level ◽

Complex Structures ◽

Ligand Complex ◽

Null Cell ◽

Protein Protein Interaction ◽

First Time

: The judicious application of ligand or binding efficiency (LE) metrics, which quantify the molecular properties required to obtain binding affinity for a drug target, is gaining traction in the selection and optimization of fragments, hits and leads. Here we report for the first time the use of LE based metric, fit quality (FQ), in virtual screening (VS) of MDM2/p53 protein-protein interaction inhibitors (PPIIs). Firstly, a Receptor-Ligand pharmacophore model was constructed on multiple MDM2/ligand complex structures to screen the library. The enrichment factor (EF) for screening was calculated based on a decoy set to define the screening threshold. Finally, 1% of the library, 335 compounds, were screened and re-filtered with the FQ metric. According to the statistical results of FQ vs activity of 156 MDM2/p53 PPIIs extracted from literatures, the cut-off was defined as FQ = 0.8. After the second round of VS, six compounds with the FQ > 0.8 were picked out for assessing their antitumor activity. At the cellular level, the six hits exhibited a good selectivity (larger than 3) against HepG2 (wt-p53) vs Hep3B (p53 null) cell lines. On the further study, the six hits exhibited an acceptable affinity (range of Ki from 102 to 103 nM) to MDM2 when comparing to Nutlin-3a. Based on our work, FQ based VS strategy could be applied to discover other PPIIs.

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Experimental pieces of evidence for Mycobacterium ulcerans dormancy

10.1101/2020.08.27.269712 ◽

2020 ◽

Author(s):

A. Loukil ◽

R. Lalaoui ◽

H. Bogreau ◽

S. Regoui ◽

M. Drancourt ◽

...

Keyword(s):

Physiological State ◽

Nile Red ◽

Buruli Ulcer ◽

Ambient Air ◽

Opportunistic Pathogen ◽

Oxygen Depletion ◽

Mycobacterium Ulcerans ◽

Ecological Niches ◽

Intracellular Lipid ◽

First Time

ABSTRACTBackgroundWhether Mycobacterium ulcerans, the etiological agent of the neglected Buruli ulcer in numerous tropical countries, would exist in a dormant state as reported for closely related Mycobacterium species, is not established.MethodologySix M. ulcerans strains were exposed to a progressive depletion in oxygen for two months, using a previously described Wayne model of dormancy; and further examined by microscopy using DDD staining, microcalorimetry and subculture in the presence of dead and replicative M. ulcerans as controls.Principal Findings/ConclusionsM. ulcerans CU001 strain died during the progressive oxygen depletion and four of five remaining strains exhibited Nile Red-stained intracellular lipid droplets after DDD staining and a 14-20-day regrowth when exposed to ambient air, diagnosing dormancy. A fifth M. ulcerans 19423 strain stained negative in DDD and slowly regrew in 27 days. Three tested M. ulcerans strains yielded microcalorimetric pattern similar to that of the negative (dead) homologous controls, differing from that of the homologous positive (replicative) controls. The relevance of these experimental observations, suggesting a previously unreported dormancy state of M. ulcerans, needs to be investigated in the natural ecological niches where M. ulcerans thrive and in Buruli ulcer lesions.Author summaryMycobacterium ulcerans is an environmental opportunistic pathogen of mammals and humans, causing a subcutaneous necrotizing infection named Buruli ulcer. Molecular detection of M. ulcerans DNA revealed different ecological niches where M. ulcerans may thrive, but the molecular biology approach does not catch the physiological state of M. ulcerans in these different ecological niches. Thus, the reservoir and the mode of transmission of M. ulcerans remain elusive. Here, we investigated experimental dormancy of M. ulcerans by using a previously described Wayne model of dormancy coupled with microscopy using DDD staining, microcalorimetry and subculture. Our findings demonstrate for the first time that some M. ulcerans strains exhibit a physiological state of dormancy; potentially limiting isolation and culture of M. ulcerans from environmental niches.

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Sequence analysis of MAP2 function in living cells

Journal of Cell Science ◽

10.1242/jcs.107.11.3115 ◽

1994 ◽

Vol 107 (11) ◽

pp. 3115-3125 ◽

Cited By ~ 5

Author(s):

J. Ferralli ◽

T. Doll ◽

A. Matus

Keyword(s):

Neuronal Cells ◽

Specific Protein ◽

Direct Result ◽

Microtubule Binding ◽

Amino Terminal ◽

Rich Domain ◽

Weak Binding ◽

Repeat Domain ◽

Combined Action ◽

Process Outgrowth

Microtubule-associated protein 2 (MAP2) is an abundant neuron-specific protein that binds to microtubules through a domain near its carboxyl terminus that contains either three or four similar repeats of a 31 amino acid motif. When expressed in non-neuronal cells by transfection MAP2 stabilises microtubules and induces their rearrangement into long bundles that are capable of supporting process outgrowth. To investigate which elements in the MAP2 sequence are involved in these functions we have constructed a series of deletion mutants of the short embryonic form of MAP2, MAP2c, and transfected them into non-neuronal cells. This showed that the strength of binding to microtubules increased with the number of repeats present in the construct. However, the repeat domain itself was insufficient for microtubule binding, which required in addition contiguous sequences either amino-terminal or carboxyl-terminal to the repeats themselves. Particularly on the amino-terminal side of the repeats, where there is a proline-rich domain, step-wise increases in the length of neighbouring sequence produced a gradual increase in microtubule binding. The apparent strength of binding to microtubules produced by mutant MAP2 forms was further correlated with the degree of bundling they induced as well as with the ability of the resulting microtubules to support process outgrowth. These results indicate that the interaction of MAP2 with microtubules is mediated by the combined action of several weak binding sites, including each of the repeat motifs and elements in the sequences on either side of them, whose additive effect produces the strong binding of the native MAP2 molecule. The results further indicate that both the bundling and stiffening of microtubules by MAP2 are correlated with the strength of its binding to them and suggest that these properties are a direct result of microtubule stabilisation.

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Development of Haemoglobin by De-embryonated Chick Blastoderms Cultured in vitro and the Effect of Abnormal RNA upon its Synthesis

Development ◽

10.1242/dev.9.1.202 ◽

1961 ◽

Vol 9 (1) ◽

pp. 202-221

Author(s):

B. R. A. O'Brien

Keyword(s):

Protein Synthesis ◽

Developmental Stages ◽

Specific Protein ◽

Cytoplasmic Protein ◽

Whole Cells ◽

Restricted Group ◽

Dividing Cells ◽

First Time ◽

Structural Code

The embryo provides a sequence of developmental stages in which proteins both structural and enzymatic appear or become detectable for the first time in a restricted group of dividing cells. The cells or tissues can be maintained in vitro for a period that may precede and include the synthesis of a specific ‘cytoplasmic’ protein. In this way systems of protein synthesis within the cells of higher organisms can be studied during those stages in which current hypotheses suggest that some structural code is passed on from the DNA of the nucleus to the cytoplasm where the synthesis of the protein becomes maximal. Acellular preparations have contributed much to the elucidation of protein synthesis, but it is doubtful whether actual net synthesis has been obtained in systems less complex than the ‘protoplast’ developed by Spiegelman (1957). In order to study the synthesis of a specific protein it seems necessary at this stage to use whole cells.

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