To quantify relative transfection rates of the plasmid, we counted the number of cells that displayed mRFP signal. As part of our TEM studies, we also observed instances where mitochondria were enclosed within membranes in infected cells, which could be indicative of mitophagy Fig. Thus, our data cumulatively indicate that mitochondrial fission is a preliminary event that subsequently contributes to apoptosis in the infected cell without having a direct impact on the establishment of a productive infection.
Our data also support the potential of mitophagy being a downstream consequence of altered mitochondrial dynamics and functionality in TC infected cells.
New World alphaviruses including VEEV are encephalitic viruses that infect humans as a result of bites by infected mosquitoes. In cases where VEEV infection leads to encephalitis, the encephalitic outcome is accompanied by death of neurons and supporting cells including astrocytes.
CPE outcomes in alphavirus infected cells may also be a consequence of transcriptional and translational inhibition induced by the capsid protein. Mitochondrial membrane potential is a cell type independent indicator of mitochondrial integrity and functional competence. In a healthy cell, membrane potential is maintained due to a functional mitochondrial respiratory chain and oxidative phosphorylation.
As a part of the energy production process, ROS is produced in normal cells as well. However, pathogenic conditions that may result in disruption of mitochondrial membrane potential will impact the integrity of the electron transport chain and result in an abnormal accumulation of ROS. Thus, a deviation in the mitochondrial membrane potential and accumulated ROS are reliable indicators of interruption of mitochondrial function.
We have observed a decrease in mitochondrial membrane potential that is also accompanied by a prominent increase in ROS in human astrocytoma cells infected with the TC strain of VEEV Fig. Notably, this phenotype was observed when cells were infected with the TC strain of VEEV, which is an attenuated strain provided as a vaccine to at-risk personnel. TC is plagued by reactogenicity concerns because it is not approved for public use.
It may be an important safety consideration to ensure that disruption of mitochondrial function is not associated with the reactogenic phenotype. It will also be of interest that the virulent strains of VEEV such as Trinidad Donkey TrD strain may result in an exaggerated oxidative stress with different kinetics in comparison to the TC strain.
Such differences in oxidative stress mechanisms may be early discriminators of virulence that establish kinetics of host inflammatory responses in early stages of post-exposure.
We observed that the production of ROS and impact of infection on mitochondrial membrane potential was lower in mosquito cells when compared to human cells in a manner that corresponded with viral load in the infected cell Fig.
This data may indicate that mosquito cells may be relatively more protected from mitochondrial dysfunction due to VEEV infection when compared to human cells. It has been demonstrated that in the case of flavivirus infections, infected insect cells show changes in antioxidant mechanisms that decrease the extent of cell death.
Mitochondrial distribution in cells and mitochondrial mobility are essential features of healthy cells.
Neuronal cells in particular are notably impacted by mitochondrial distribution between the cell body and axon. Anterograde transport of mitochondria in neurons is mediated by kinesin motors on microtubules, while retrograde transport is mediated by dynein motors.
In HSV infections, it was demonstrated that anterograde transport of mitochondria was disrupted due to impaired association of mitochondria with the kinesin motor. Mitochondrial congregation in a perinuclear manner has been indicated to correspond with oxidative stress in many neurodegenerative states.
In the context of neurons, this will have a direct impact on neuronal functionality, which may also contribute to neuronal death and encephalitis in VEEV infection. Viral protein localization in infected cells is a well-documented process for many viruses that directly influence innate immune responses of the host. Our preliminary studies have suggested that while transfection of a plasmid that encodes VEEV capsid can produce similar phenotypes in astrocytoma cells, the extent of mitochondrial dysfunction is lesser than that observed in infected cells, indicating that multiple viral components may contribute to the observed mitochondrial events data not shown.
Similar outcomes were demonstrated for HCV where the viral core protein was shown to indicate with host enzymatic components that localized to mitochondria. Of interest to us was the observation that the host kinase PINK1 and ubiquitin ligase Parkin localized in mitochondria of infected cells Figs. PINK1 and Parkin have generated significant interest in recent years for their association with mitochondrial dysfunction in neurodegeneration.
Of direct relevance to mitochondrial motility, PINK1 activity is known to phosphorylate Miro, a connecting link between mitochondria and kinesin. In addition, Parkin ubiquitinates Miro, which ultimately leads to its degradation.
PINK1 is also known to phosphorylate Parkin and contribute to its ubiquitination function. Indeed, this has been demonstrated in the case of Rubella virus capsid protein, a post-translationally modified viral core protein, specifically the phosphorylated form, associated with mitochondrial p32 protein. Such indications as described thus far usually lead to downstream compensatory mechanisms initiated by the host cell, directed toward removing damaged mitochondria.
This is an integral part of maintaining cell survival while the host contends with cellular stress. These mechanisms may include mitochondrial fission or fusion, in which damaged parts of mitochondria are excised and smaller mitochondria fuse to create larger mitochondria. In the context of HCV infection, it has been demonstrated that mitochondrial localization of phosphorylated form of Drp1 protein Drp1-Ser was a prelude to mitochondrial fission. Our observation that phosphorylated Drp1-Ser localized to mitochondria in a transient manner is suggestive of a dynamic relationship between Drp1 localization, mitochondrial fission and VEEV infection.
Transmission electron microscopic analysis of TC infected cells showed changes in mitochondrial structure Fig. In a recent publication on mitochondrial changes induced in parvoviral infection, the authors include similar observations on mitochondrial dysfunction including drop in membrane potential and accumulation of ROS as we have indicated here. The authors provide electron microscopic visuals of abnormal mitochondrial phenotypes that are similar to the ones we have included in our results including heterogeneously swollen mitochondria, membrane blebbing and disappearance of cristae.
Our studies led us to question whether mitochondrial fission was relevant to viral multiplication. Our studies with the inhibitor Mdivi-1 indicated that mitochondrial fission does not contribute to viral load in infected cells Fig.
It has been reported that viruses do, in fact, utilize the ROS rich environment of infected cells in a manner that may favor aspects of viral multiplication including RNA synthesis. In the case of flaviviruses, it was shown that the guanylyltransferase activity of NS5 encoded polymerase was enhanced by oxidative conditions. Our follow up hypothesis was that mitochondrial fission may contribute to the ensuing apoptosis in infected cells.
Our TEM studies revealed cellular morphology that was consistent with exaggerated membrane blebbing and apoptosis. When treated with an inhibitor of mitochondrial fission, we observed a statistically significant reduction in caspase cleavage, thus adding support to our suggestion that mitochondrial fission was a contributor to apoptosis of VEEV-infected cells.
Mitophagy is a mitochondrial elimination process in which defective mitochondria are enveloped in lipid bilayers to form autophagosomes that contain whole mitochondria. Such mitochondrial engulfment by the phagosomes leads to selective elimination of damaged mitochondria, thus decreasing the mitochondrial dysfunction load as a means of restoring cellular health.
We observed that mitochondrial alterations in VEEV-infected cells contributed to mitophagic elimination of mitochondria. In our TEM images, we also observed that there were multiple mitochondria in infected cells that were associated with encircling membranes. Taken together, our data suggest that VEEV infection produces a significant impact on mitochondrial dynamics in infected cells.
The onset of mitochondrial dysfunction during early stages of VEEV infection may set the stage for downstream events that culminate in neuronal death. Mitochondrial dysfunction may be associated in a cause and effect manner with pronounced alterations to the mitochondrial proteome; a deeper understanding of which will shed more light on the mitochondrial influence on disease progression in New World alphavirus infections. Notably, mitochondria as a therapeutic target is being explored for different neurodegenerative disorders including optic neurodegeneration.
TC attenuation from the fully virulent Trinidad Donkey TrD strain has been previously characterized. Cells were seeded in either a well plate or an 8-well chamber slide, at the indicated concentration for each assay, in order to attain confluency within 24 hours. The media was removed and saved, and thereafter referred to as conditioned media.
The viral inoculum was then removed and replaced with the conditioned media. Plaque assays were performed as previously described. TMRE is a cell-permeable reagent that preferentially accumulates in active mitochondria. TMRE experimental controls included carbonyl cyanide 4- trifluoromethoxy phenylhydrazone FCCP Abcam , a potent mitochondrial oxidative phosphorylation un-coupler, and RVFV, a virus previously shown to induce mitochondrial stress.
U87MG cells were seeded at 10, cells per well in a black well plate. Mitochondrial membrane and cytosolic fractions were obtained using the Mitochondrial Extraction Kit for Cultured Cells Thermo Scientific according to the manufacturer's instructions. Preparation of whole cell lysates and western blot have been previously described.
Transfections were performed using Attractene Transfection Reagent Qiagen as per the manufacturer's instructions. Siddiqui University of California, San Diego , which was generated as previously described. Immunofluorescence assays were performed as previously described. Fluorescent secondary antibodies labeled with Alexa Fluor and Thermo Fisher were used to visualize proteins of interest. After infection, samples were washed three times with 0.
Subsequently, samples were fixed with 2. The fixative was replaced with a 0. Samples were then washed with ultra-pure water three times. The samples were then treated with propyleneoxide for 1 hour and infiltrated in a mixture of propyleneoxide and TAAB Epon.
Mitochondrial images were obtained at a magnification of 25,X, while whole cell images were obtained at a magnification of 2,X. Mdivi-1 was dissolved in dimethyl sulfoxide DMSO prior to dilution in complete media. U87MG cells were pre-treated with either 0. Unless otherwise stated, graphs and images are the average of three biologically independent experiments. Standard deviations were calculated using Microsoft Excel and represented where applicable. Statistical significance was tested by unpaired, two-tailed Student's t-test between the sample data and time-matched control.
The mitophagy reporter plasmid pAT was generously provided by Dr. Aleem Siddiqui. The microscopy studies were supported by CSM start-up fund The phosphorylated Bad antibodies were a kindly provided by Dr. From Oxford University. The lead researcher in the person clinical trial, Dr. It is widely recognized that mitochondrial dysfunction may contribute to the profound fatigue associated with this condition. Why else would a company do the lab studies, do the type II diabetes study, begin a several hundred-person study of the most severe form of fatty liver disease and then start a placebo-controlled study in long COVID?
Why would a researcher like Rathman spend her time with it? Of course, time will tell. Please, not to be a wet blanket but really? All of this looks like what you see in retail over the counter I have. Are they just going to make money with a special name put on it. It is already none a certain combo of amino acids are very helpful even from info on this site I do not have hope for this, sorry, just my opinion. What happens if the trial does succeed and people with long COVID show a small but still significant increase in energy which is linked to an increase in ATP production and improved muscle metabolism.
That provides evidence that energy production rather than depression, malingering, or whatever is a problem in long COVID — pointing researchers in that possibly very fruitful direction. No, but a good clinical trial outcome would move the field in a certain way.
Neuroinflammation would probably get more attention. Oxidative stress certainly would as well doctors would know that NAC at certain doses can be helpful. That would probably reorient them a bit. Even if they are studying corn syrup. That of course assumes PEM is happening in long-Covid. If I have it right they are assessing phosphocreatine? That appears to measure how quickly the mitochondria recover after being stressed.
Anecdotally, I always felt a much more improvement to my brain fog from glutatione IVs than ever from taking oral glutathione even liposomal or NAC. Oral supplements have always come with problematic side effects for me.
That would lend itself to further exploration in the gut microbiome avenue. I had my DNA sequenced back when Courtagen was looking into these things. Eur J Immunol. Ultrastructural characterization of zika virus replication factories. Cell Rep. Sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.
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Hence, the length of a leaf cell above is one order of magnitude larger than the diameter of a red blood cell. When comparing the size of a bacterium with the HIV virus, the values given are in different units. The different units have to be converted so that they are the same.
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