Biological Function And Regulating Mechanism Of SIRT1after Traumatic Brain Injury

Background: Traumatic brain injury is a common central nervous system disease andfrequently leads to neurological dysfunction or death. The mechanisms that trigger TBIare complicated, including glutamate-mediated excitotoxicity, generation of reactiveoxygen species, DNA damage, regulation of pro-apoptotic factors, and so on. Currently,there are less neuroprotectants for TBI in clinic as the cellular metabolic networks afterTBI are not known very well. To fnd a key molecule, which can activate endogenoussurvival signaling and repair mechanism, is a critical strategy for TBI therapy.Sir2is an NAD-dependent deacetylase that connects metabolism with longevity inyeast. Mammals have seven sirtuins paralogs (SIRT1-7), with SIRT1being the closest tothe yeast Sir2. Moreover, SIRT1is the best-characterized sirtuin among the seven andexpressed at a high level in the brain compared to other organs. Manipulation of SIRT1’sactivity or levels by pharmacological and genetic means in several models ofneurodegenerative diseases demonstrated its neuroprotective credentials. However, recent data have indicated that under certain contexts, SIRT1inhibition, rather thanactivation, is neuroprotective. The contradictory results seemingly imply that SIRT1might play a detrimental role in such conditions. The inconsistencies highlight thecomplex nature of SIRT1-mediated effects. Thus, an understanding of the function ofSIRT1in TBI is necessary to design effective therapeutic strategies.Purpose:(1) to elucidate the variation and function of SIRT1in TBI;(2) to explore theinteractions between SIRT1and MAPK/ERK in TBI;(3) to explore the potentialregulation mechanism between SIRT1and homer1a.Methods:(1) Cell culture: cortical neurons culture from embryo mice;(2) Model:mechanical neuronal injury model and mouse closed head injury model induced by aweight-drop device;(3) Expression and location of molecule: Western blot,immunofluorescence and immunohistochemistry;(4) Neuronal injury assessment: LDHassay and PI/Hoechst staining;(5) Brain injury assessment: neurological severity score;(6) Apoptosis: detection of caspase-3activation by Western blot and TUNEL staining;(7)Regulation of SIRT1: down-regulation of SIRT1by inhibitor salermide and siRNA andup-regulation of SIRT1by activator resveratrol;(8) Interference of ERK/MAPK pathway:ERK inhibitor PD98059and U0126;(9) Regulation of Homer1a: up-regulation ofHomer1a by transfection primary neurons with lentivirus vector.Results:Chapter one: In mechanical neuronal injury model, compared to normal neurons, theLDH release was signifcantly increased in neurons within30min of the start of scratchinjury. The TUNEL positive neurons were signifcantly increased in24h group afterinjury compared to normal neurons. The expression of cleaved Caspase-3wassignifcantly elevated from30min and decreased from24h. These results indicated thatthe scratch model could induce apoptotic cell death in primary cortical neurons. In themouse closed head injury model, compared to normal control group, the level of bloodglucose was signifcantly increased at3h after TBI and reached the peak level at24hafter trauma. Then, the level of blood glucose was gradually decreased to normal level at5days after TBI. The number of TUNEL-positive neurons in the injured-side cortex wassignifcantly increased at12h after TBI compared to the controls. The expression ofcleaved Caspase-3in injured-side cortex was signifcantly elevated from3h and lastedfor3days after TBI and then decreased to normal level. These results indicated that the weight-drop model could induce neuronal apoptosis in injured-side cortex. Moreover, theprotein level of SIRT1was signifcantly increased after trauma and then decreased tonormal level both in primary cortical neurons and injured-side cortex. In addition, innormal adult mouse brain, there were a few SIRT1-positive neurons both in cortex andhippocampus; However, SIRT1was highly expressed in neurons of injured-side cortexand almost exclusively retained in cytoplasm, while the SIRT1-positive neurons in CA1region of injured-side hippocampus showed a slight increase compared to hippocampusof normal mice.Chapter two: The further study showed that MAPK/ERK pathway was activated aftertraumatic brain injury in vitro and in vivo. In mechanical neuronal injury model, neuronspretreated with25,50or100μM salermide for24h appeared signifcantly decreasingcell survival. The LDH release signifcantly increased in neurons pretreated with50μMand100μM salermide but not in25μM. When endogenous SIRT1was signifcantlyinhibited after scratch, the expressions of cleaved Caspase-3were signifcantly elevatedand the activation of MAPK/ERK pathway was signifcantly inhibited in neuronspretreated with50or100μM salermide and showed a concentration-dependent way.Consistent with the results caused by salermide, the expression of cleaved Caspase-3andthe activation of MAPK/ERK pathway showed similar tendencies in neurons transfectedSIRT1siRNA. The SIRT1inhibitor salermide (200or400μMsalermide in2μL volumes)was injected into the lateral ventricles of the mouse brain1h before TBI. The shamgroup was injected withequal volumes of DMSO. Both200and400μM salermide couldinhibit the SIRT1elevation after TBI. When the expression of SIRT1was inhibited, ERKactivation was signifcantly reduced in mice after TBI.To determine whether the activation of MAPK/ERK pathway could regulate the levelofendogenous SIRT1, neurons were pretreated withthe20μM PD98059for1hand thenexposed to scratch injury for1h. when the ERK activation was inhibited by PD98059,the expression of SIRT1was signifcantly decreased. The expression of cleavedCaspase-3in neurons pretreated with PD98059was signifcantly decreased. TUNELstaining showed that the number of apoptotic neurons pretreated with PD98059wassignifcantly lower than neurons pretreated with DMSO. When the ERK activation wasinhibited, the expression of SIRT1was signifcantly decreased in mice injected withPD98059or U0126, which is consistent with the results in vitro. In addition, inhibition ofthe activation of MAPK/ERK pathway could signifcantly reduced the level of blood glucose and improved the NSS of mice after TBI; However, inhibited SIRT1couldsignifcantly increased the level of blood glucose and aggravated the NSS of mice afterTBI.Chapter three: The expression of Homer1a was fluctuant but the expressionHomer1b/c was stable after traumatic brain injury in vitro and in vivo. Homer1a andp-ERK were mainly distributed in cytoplasm and axons of neurons. Inhibited SIRT1withsiRNAdecreased the expressionof homer1a and β-cateninof neurons after scratch injury.However, activated SIRT1by resveratrol increased the levels of homer1a and β-cateninof neurons. Additionally, inhibition of the activation of MAPK/ERK pathway couldsignifcantly reduced the expression of homer1a in vitro and in vivo. Up-regulation ofHomer1a by transfection with lentivirus vector decreased the number of neurons ofTUNEL positive staining, increased the expression of β-catenin, and inhibited theexpressions of SIRT1and p-ERK, suggested that exogenous homer1a might negativelyregulate the expression of SIRT1in TBI.Conclusion: The data in present study demostrate that SIRT1and Homer1a, asendogenous protective molecules, have neuroprotective effects against traumatic braininjury. SIRT1might regulate the expression of homer1a by interacting with MAPK/ERKpathway and β-catenin, and exogenous homer1a might regulate the expression of SIRT1by negative feedback in TBI. The present study not only elucidates a self-protectivemechanism in neurons, but also provides potential pharmacological targets and novelstrategies of treatment for traumatic brain injury.