Effects of Traumatic Brain Injury Variability on Autophagy Regulation

Gabriel Peterman Author

07/30/2021 Added

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A video describing research from the Kievit lab focusing on how brain injury severity affects autophagy - a mechanism by which cells degrade their waste products.

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[00:00:07.730]Humans,... We love to eat. Whether because we're hungry, stressed,
[00:00:12.980]or just plain bored.
[00:00:14.540]Eating has been the cornerstone of modern society since the beginning.
[00:00:21.590]Hi.
[00:00:22.340]I'm Gabriel Peterman with the Keivit lab.
[00:00:24.350]here at the university of Nebraska Lincoln. Did you know that, like you,
[00:00:28.730]your cells also liked to eat? Through a mechanism termed autophagy,
[00:00:32.420]cells can recycle their nutrients by eating their inner components. In one
[00:00:37.280]form of brain trauma,
[00:00:38.480]called traumatic brain injury. Autophagy appears to be impeded following tissue
[00:00:43.190]damage. In this video,
[00:00:45.290]I'm going to describe to you the mechanisms behind autophagy
[00:00:48.300]Explain to you
[00:00:49.280]the role of autophagy as a potential therapeutic target for treating traumatic
[00:00:52.850]brain injury, and present new experimental results to you
[00:00:56.570]showing how autophagy is affected by a variety of brain injury severities.
[00:01:02.060]Autophagy kicks off when a cell experiences stress,
[00:01:05.300]whether this stress be caused by nutritional deficiencies
[00:01:09.380]or harful conditions within the internal environment.
[00:01:11.600]In this example, stress is caused by a dysfunctional mitochondria discharging
[00:01:16.220]dangerous reactive oxygen species, a sight
[00:01:19.430]all too common in brain tissue after injury.
[00:01:22.430]This stressful environment signals the activation of the autophagic biochemical
[00:01:26.930]pathway,
[00:01:27.920]which eventually activates a complex that contains the Beclin-1 protein.
[00:01:32.330]This Beclin-I complex then goes on to initiate the formation of the
[00:01:36.800]phagophoric membrane,
[00:01:37.970]and also the lipidation of the LC3-I into LC3-II,
[00:01:42.230]which acts as an important
[00:01:43.910]phagophore receptor protein. After cellular pathways
[00:01:48.200]tag damage components and other unwanted debris with the ubiquitin protein.
[00:01:54.050]Autophagy cargo proteins,
[00:01:56.980]including p62 will carry the autophagy target back to the phagophpric
[00:02:02.030]membrane to bond with LC3-II. Next,
[00:02:05.480]the phagophore will mature around the autophagy target and create an
[00:02:09.080]autophagosome once fully formed.
[00:02:11.150]This autophagosome will shuttle the target to a cell's
[00:02:14.840]lysosome. A lysosome is a cellular organelle that contains multiple
[00:02:19.700]degrading enzymes. Once close enough,
[00:02:22.970]the lysosome will begin fusing with the autophagosome to create an auto-
[00:02:26.780]lysosome. After the autophagy target is fully degraded,
[00:02:31.190]Its leftovers can be recycled back to the cell for nutrients.
[00:02:35.210]Traumatic brain injury is a form of neurological trauma
[00:02:38.390]that makes up one third of all injury- related deaths in the United States.
[00:02:43.160]After suffering from the primary injury victims often suffer the spread of
[00:02:47.690]secondary damage throughout the brain tissue,
[00:02:50.150]mostly in the form of neuro- inflammation and oxidative stress.
[00:02:54.440]One of the troubling observations associated with some brain injuries is the
[00:02:59.140]accumulation of damage cellular components within the neural tissue,
[00:03:03.160]such as damaged mitochondria and aggregated proteins,
[00:03:06.440]which have been linked to other degenerative diseases
[00:03:09.516]such as Alzheimer's and Parkinson's disease.
[00:03:12.250]Autophagy is normally the means by which cells get rid of these damaged
[00:03:16.010]components. However,
[00:03:17.860]the presence of these damaged components suggests that autophagy can be impaired
[00:03:22.066]following brain trauma. Therefore,
[00:03:24.250]autophagy is a potential therapeutic target for treating traumatic brain injury.
[00:03:29.984]However, before treatments can be developed,
[00:03:32.654]the mechanisms of normal autophagy that are affected by traumatic brain injury
[00:03:37.000]must be understood. Here at the Kievit lab,
[00:03:40.355]we are working to explain a autophagy in relation to brain injury.
[00:03:44.140]Now there have been some past studies focusing on how traumatic brain injury
[00:03:48.703]affects autophagy.
[00:03:50.710]Although, many of these past experiments focus on severe brain injuries.
[00:03:55.150]However,
[00:03:56.213]over 80% of all clinical traumatic brain injuries are mild, resulting from
[00:04:01.163]simple things such as sports concussions. Therefore,
[00:04:04.673]our study will differ in that we will be comparing a autophagy differences after a
[00:04:08.753]variety of injury severities ranging from mild to severe.
[00:04:13.220]In this project,
[00:04:14.240]we hypothesized that the mechanisms of autophagy in neural tissue will become
[00:04:19.097]increasingly impaired as the severity of traumatic brain injury increases
[00:04:24.580]to fulfill our experiment.
[00:04:26.170]We employed three different controlled cortical impact mouse models of brain
[00:04:29.890]injury with the device shown. The first traditional injury model involves cutting
[00:04:34.330]open a mouse's skull and directly impacting the
[00:04:37.240]mouse's brain with a metal rod producing a very severe injury.
[00:04:41.156]The second silicone injury model was similar to the traditional model. However,
[00:04:46.180]the mouse's brain was impacted with a soft silicone gel tip instead of a hard
[00:04:50.607]metal tip at a much lower speed, producing mild brain trauma.
[00:04:54.250]The final helmet injury model involves impacting a helmet on top of the mouse's
[00:04:59.399]head instead of directly hitting the mouse's brain.
[00:05:02.494]The mouse also sat on a foam pad.
[00:05:05.196]This helmet model produced a diffuse injury and injury severity was varied by
[00:05:10.305]varying the remaining distance traveled by the impactor tip after contacting
[00:05:14.345]the mouse's helmet. Next,
[00:05:16.558]we took tissue samples from the mice brains three days after injury. Finally,
[00:05:21.030]we analyzed the following autophagy related protein levels using the Western blot
[00:05:25.840]method by observing increases or decreases in these protein levels.
[00:05:30.521]We can make inferences about the mechanisms of autophagy affected by traumatic
[00:05:34.811]brain injury. Based on the proteins' known functions, here are our results:
[00:05:39.554]We quantified autophagy proteins,
[00:05:41.869]according to the optical densities of their Western blots
[00:05:44.650]normalized to beta actin. In the left brain cortex, where the injury occurred,
[00:05:48.660]the observed increase of Beclin-1.
[00:05:50.890]suggests that the autophagy pathway is upregulated following severe brain
[00:05:54.240]injuries and the elevation of ATG7 and ATG3 levels suggest an
[00:05:58.353]increase of phagophore formation. The right cortex samples mirror this result.
[00:06:02.876]However,
[00:06:03.850]if autophagy was working normally we would expect to see p62 levels
[00:06:06.990]decrease from the control as p62 is degraded with its target
[00:06:10.077]during normal autophagy. Therefore, autophagy appears to be impeded in the severe
[00:06:14.083]controled cortical impact (CCI) models due to increases of p62 levels. The decrease of all
[00:06:18.284]proteins, including p62,
[00:06:20.503]in the mild silicone model implies that autophagy is not being amplified
[00:06:23.860]following injury. However, the rate of autophagy is not impaired.
[00:06:26.984]Although variability and error are high in the cortex samples,
[00:06:30.798]the hippocampus samples, where injury damage is expected to spread mostly through
[00:06:34.989]neuroinflammation and oxidative stress, contain more concrete protein
[00:06:38.411]differences that strengthen our claims. In both hippocampus regions,
[00:06:42.310]all protein levels are visibly increased in the severe CCI model from both the
[00:06:45.944]control and mild injury models. Finally,
[00:06:48.538]we come to our helmeted diffuse models where we varried the distance traveled
[00:06:51.630]after impact at 3, 9, and 15 millimeters. Surprisingly the least,
[00:06:55.419]and most severe 3 and 15 mm models appear to have lower levels of
[00:06:58.910]autophagy activation than the medium severity model. However,
[00:07:03.021]we do see a trend in the hippocampus p62 data where autophagy impairment is
[00:07:07.010]highest in the most severe model and lowest in the least severe model.
[00:07:11.876]Our results suggested that autophagy is activated but impaired after severe
[00:07:15.926]traumatic brain injuries.
[00:07:17.556]And that autophagy is functional yet downregulated in milder injuries.
[00:07:21.646]Therefore,
[00:07:22.743]autophagy modification would offer an effective strategy for reducing the
[00:07:26.120]secondary injuries associated with brain injury.
[00:07:29.240]Future work will include confirming our results through increasing our Western
[00:07:32.640]blot sample size and observing autophagosome accumulation with confocal imaging.
[00:07:37.780]We will also investigate how the reduction of reactive oxygen species through
[00:07:42.600]antioxidant nanoparticles will impact autophagy.
[00:07:45.621]We hope that our work here provides a basis for other researchers to design
[00:07:49.335]treatments for brain injuries,
[00:07:51.530]especially in relation to autophagy I'm Gabriel Peterman signing off.
[00:07:55.109]Thank you for watching!!! ;)

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Effects of Traumatic Brain Injury Variability on Autophagy Regulation

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