AUTOPHAGY 2016, VOL. 12, NO. 11, 2256–2257 http://dx.doi.org/10.1080/15548627.2016.1221565
AUTOPHAGIC PUNCTUM
Brain trauma and autophagy: What flies and mice can teach us about conserved responses Eric P. Ratliffa,b, Ayeh Barekata,b, Marta M. Lipinskic, and Kim D. Finleya,b a Donald P. Shiley BioScience Center, San Diego State University, San Diego, CA, USA; bDepartment of Biology, San Diego State University, San Diego, CA, USA; cShock, Trauma, and Anesthesiology Research (STAR) Center, Department of Anesthesiology, University of Maryland School of Medicine, Baltimore, MD, USA
ABSTRACT
Drosophila models have been successfully used to identify many genetic components that affect neurodegenerative disorders. Recently, there has been a growing interest in identifying innate and environmental factors that influence the individual outcomes following traumatic brain injury (TBI). This includes both severe TBI and more subtle, mild TBI (mTBI), which is common in people playing contact sports. Autophagy, as a clearance pathway, exerts protective effects in multiple neurological disease models. In a recent publication, we highlighted the development of a novel repetitive mTBI system using Drosophila, which recapitulates several phenotypes associated with trauma in mammalian models. In particular, flies subjected to mTBI exhibit an acute impairment of the macroautophagy/autophagy pathway that is restored 1 wk following traumatic injury exposure. These phenotypes closely resemble temporary autophagy defects observed in a mouse TBI model. Through these studies, we also identified methods to directly assess autophagic responses in the fly nervous system and laid the groundwork for future studies designed to identify genetic, epigenetic and environmental factors that have an impact on TBI outcomes.
One of the major causes of human morbidity and mortality worldwide involves traumatic brain injury (TBI). Along with severe TBI, repetitive mild traumatic brain injuries (mTBI) are now known to result in both acute and long-term deficits to the nervous system. In normal environments organisms are routinely exposed to a wide range of stressful conditions and have evolved diverse mechanisms that mitigate damage and promote the repair of cells and tissues. Unfortunately, relatively little is still known about the factors regulating organismal responses to TBI. Confounding our understanding of these trauma-related factors has been the lack of an effective genetic system to model conserved tissue-specific and pathway responses. There has been growing interest in the role that intracellular clearance mechanisms, such as autophagy, have on longterm neural function following TBI. Drosophila genetics has been instrumental in examining the role of autophagy in the maintenance of the nervous system and in neurodegenerative disorders. We have recently reported on a repetitive mTBI model optimized for adult Drosophila. mTBI-treated flies exhibit many phenotypes associated with TBI in humans and mammalian models, including sleep-related behavioral defects, activation of neuroinflammatory responses and increased phosphorylation of the human MAPT/Tau protein in the brain. We also developed novel methodologies to assess
ARTICLE HISTORY
Received 20 July 2016 Revised 1 August 2016 Accepted 1 August 2016 KEYWORDS
Atg8; autophagy; brain trauma; conserved responses; Drosophila; LC3; lysosomal defects; mouse; p62; Ref(2)P
autophagy in mTBI-treated flies. Using an anti-GABARAP antibody that recognizes the endogenous fly Atg8a protein, the number of positive puncta forming in adult neurons was counted in whole brains. Twenty-four hours following injury, mTBI-treated flies exhibit a significant increase in the number of autophagosomes in neuronal cells. These data are consistent with a previous Autophagy report (Sarkar, et al.), wherein GFP-LC3 transgenic mice show an acute accumulation of autophagic puncta 24 h following controlled cortical impact TBI. The buildup of autophagsomes is caused by the inhibition of autophagic flux, as reflected by the increase in ubiquitinated proteins and SQSTM1/p62, and not through the activation of upstream signaling kinases, such as ULK1. This buildup is accompanied by, and most likely due to, lysosomal impairments after TBI. Consistent with these findings, we observed a significant increase in the fly SQSTM1 homolog Ref(2)P, ubiquitinated proteins, and Atg8a-II levels in the fly brains following mTBI exposure. After the acute inhibition of the autophagy pathway, both studies presented data suggesting that autophagic flux was resolved one wk following injury. For example, neural ubiquitinated and Ref(2)P protein profiles in mTBI-treated flies resolve to levels observed in uninjured controls, even though ref(2)P expression is increased at this time. Similarly, the levels of ubiquitinated proteins and SQSTM1 decrease to nearly that
CONTACT Kim D. Finley kfi[email protected] Donald P. Shiley BioScience Center, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-4650. Punctum to: Barekat A, Gonzalez A, Mauntz RE, Kotzebue RW, Molina B, El-Mecharrafie N, Conner CJ, Garza S, Melkani GC, Joiner WJ, Lipinski MM, Finley KD, Ratliff EP. Using Drosophila as an integrated model to study mild repetitive traumatic brain injury. Sci Rep 2016; 6:25252. PMID: 27143646; http://dx.doi.org/10.1038/srep25252. © 2016 Taylor & Francis
AUTOPHAGY
of controls 1 wk following TBI in mice. Flies also show a decrease in Atg8a protein levels 1 wk after mTBI, although Atg8a mRNA expression is largely unchanged between control and injured fly cohorts. Furthermore, Drosophila neural tissues show an increase in Atg8a-II:Atg8a-I ratios following TBI. We surmised that newly synthesized Atg8a-I protein is rapidly cleaved, incorporated into phagophores and degraded in the lysosome, strongly suggesting an increase in autophagic flux. These studies are the first to our knowledge to adapt the western blot protocol in adult Drosophila neural tissue to examine both acute and protracted alterations in the autophagy pathway. Further, these data highlight the utility of using the Atg8a-II:Atg8a-I ratio comparisons when examining Drosophila neural tissue. The molecular mediators responsible for restoring autophagy 1 wk post-TBI require further examination. Upregulation of lysosomal markers is observed in the mouse controlled cortical impact model suggesting that lysosomal biogenesis is increased.
2257
Given the similar findings between our Drosophila and mouse TBI models, we postulate that the genetic power of the Drosophila model system can be used to identify key autophagy components that promote neuronal health and survival following mTBI, which can then be confirmed in the mammalian trauma models. Indeed, the flexibility of this mTBI system and the rapid aging of adult flies permit the long-term assessment of neural trauma responses. Our data demonstrate that profound differences in mTBI responses occur between young (1-wk) and middle-aged flies (3-wk). Another advantage of our fly model, along with the ability to conduct standard genetic experiments, is that Drosophila reagents readily permit tissue and cell-type specific knockdown and overexpression studies of select genes. In summary, our goal is to utilize Drosophila to further highlight the role of autophagy in acute and long-term TBI-related studies and to generate unique insights into neurodegenerative disorders, such as Alzheimer’s disease and chronic traumatic encephalopathy.
AUTOPHAGIC PUNCTUM
Brain trauma and autophagy: What flies and mice can teach us about conserved responses Eric P. Ratliffa,b, Ayeh Barekata,b, Marta M. Lipinskic, and Kim D. Finleya,b a Donald P. Shiley BioScience Center, San Diego State University, San Diego, CA, USA; bDepartment of Biology, San Diego State University, San Diego, CA, USA; cShock, Trauma, and Anesthesiology Research (STAR) Center, Department of Anesthesiology, University of Maryland School of Medicine, Baltimore, MD, USA
ABSTRACT
Drosophila models have been successfully used to identify many genetic components that affect neurodegenerative disorders. Recently, there has been a growing interest in identifying innate and environmental factors that influence the individual outcomes following traumatic brain injury (TBI). This includes both severe TBI and more subtle, mild TBI (mTBI), which is common in people playing contact sports. Autophagy, as a clearance pathway, exerts protective effects in multiple neurological disease models. In a recent publication, we highlighted the development of a novel repetitive mTBI system using Drosophila, which recapitulates several phenotypes associated with trauma in mammalian models. In particular, flies subjected to mTBI exhibit an acute impairment of the macroautophagy/autophagy pathway that is restored 1 wk following traumatic injury exposure. These phenotypes closely resemble temporary autophagy defects observed in a mouse TBI model. Through these studies, we also identified methods to directly assess autophagic responses in the fly nervous system and laid the groundwork for future studies designed to identify genetic, epigenetic and environmental factors that have an impact on TBI outcomes.
One of the major causes of human morbidity and mortality worldwide involves traumatic brain injury (TBI). Along with severe TBI, repetitive mild traumatic brain injuries (mTBI) are now known to result in both acute and long-term deficits to the nervous system. In normal environments organisms are routinely exposed to a wide range of stressful conditions and have evolved diverse mechanisms that mitigate damage and promote the repair of cells and tissues. Unfortunately, relatively little is still known about the factors regulating organismal responses to TBI. Confounding our understanding of these trauma-related factors has been the lack of an effective genetic system to model conserved tissue-specific and pathway responses. There has been growing interest in the role that intracellular clearance mechanisms, such as autophagy, have on longterm neural function following TBI. Drosophila genetics has been instrumental in examining the role of autophagy in the maintenance of the nervous system and in neurodegenerative disorders. We have recently reported on a repetitive mTBI model optimized for adult Drosophila. mTBI-treated flies exhibit many phenotypes associated with TBI in humans and mammalian models, including sleep-related behavioral defects, activation of neuroinflammatory responses and increased phosphorylation of the human MAPT/Tau protein in the brain. We also developed novel methodologies to assess
ARTICLE HISTORY
Received 20 July 2016 Revised 1 August 2016 Accepted 1 August 2016 KEYWORDS
Atg8; autophagy; brain trauma; conserved responses; Drosophila; LC3; lysosomal defects; mouse; p62; Ref(2)P
autophagy in mTBI-treated flies. Using an anti-GABARAP antibody that recognizes the endogenous fly Atg8a protein, the number of positive puncta forming in adult neurons was counted in whole brains. Twenty-four hours following injury, mTBI-treated flies exhibit a significant increase in the number of autophagosomes in neuronal cells. These data are consistent with a previous Autophagy report (Sarkar, et al.), wherein GFP-LC3 transgenic mice show an acute accumulation of autophagic puncta 24 h following controlled cortical impact TBI. The buildup of autophagsomes is caused by the inhibition of autophagic flux, as reflected by the increase in ubiquitinated proteins and SQSTM1/p62, and not through the activation of upstream signaling kinases, such as ULK1. This buildup is accompanied by, and most likely due to, lysosomal impairments after TBI. Consistent with these findings, we observed a significant increase in the fly SQSTM1 homolog Ref(2)P, ubiquitinated proteins, and Atg8a-II levels in the fly brains following mTBI exposure. After the acute inhibition of the autophagy pathway, both studies presented data suggesting that autophagic flux was resolved one wk following injury. For example, neural ubiquitinated and Ref(2)P protein profiles in mTBI-treated flies resolve to levels observed in uninjured controls, even though ref(2)P expression is increased at this time. Similarly, the levels of ubiquitinated proteins and SQSTM1 decrease to nearly that
CONTACT Kim D. Finley kfi[email protected] Donald P. Shiley BioScience Center, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-4650. Punctum to: Barekat A, Gonzalez A, Mauntz RE, Kotzebue RW, Molina B, El-Mecharrafie N, Conner CJ, Garza S, Melkani GC, Joiner WJ, Lipinski MM, Finley KD, Ratliff EP. Using Drosophila as an integrated model to study mild repetitive traumatic brain injury. Sci Rep 2016; 6:25252. PMID: 27143646; http://dx.doi.org/10.1038/srep25252. © 2016 Taylor & Francis
AUTOPHAGY
of controls 1 wk following TBI in mice. Flies also show a decrease in Atg8a protein levels 1 wk after mTBI, although Atg8a mRNA expression is largely unchanged between control and injured fly cohorts. Furthermore, Drosophila neural tissues show an increase in Atg8a-II:Atg8a-I ratios following TBI. We surmised that newly synthesized Atg8a-I protein is rapidly cleaved, incorporated into phagophores and degraded in the lysosome, strongly suggesting an increase in autophagic flux. These studies are the first to our knowledge to adapt the western blot protocol in adult Drosophila neural tissue to examine both acute and protracted alterations in the autophagy pathway. Further, these data highlight the utility of using the Atg8a-II:Atg8a-I ratio comparisons when examining Drosophila neural tissue. The molecular mediators responsible for restoring autophagy 1 wk post-TBI require further examination. Upregulation of lysosomal markers is observed in the mouse controlled cortical impact model suggesting that lysosomal biogenesis is increased.
2257
Given the similar findings between our Drosophila and mouse TBI models, we postulate that the genetic power of the Drosophila model system can be used to identify key autophagy components that promote neuronal health and survival following mTBI, which can then be confirmed in the mammalian trauma models. Indeed, the flexibility of this mTBI system and the rapid aging of adult flies permit the long-term assessment of neural trauma responses. Our data demonstrate that profound differences in mTBI responses occur between young (1-wk) and middle-aged flies (3-wk). Another advantage of our fly model, along with the ability to conduct standard genetic experiments, is that Drosophila reagents readily permit tissue and cell-type specific knockdown and overexpression studies of select genes. In summary, our goal is to utilize Drosophila to further highlight the role of autophagy in acute and long-term TBI-related studies and to generate unique insights into neurodegenerative disorders, such as Alzheimer’s disease and chronic traumatic encephalopathy.
