Neuroprotective Effect Of Histone Deacetylase Inhibitor On Traumatic Brain Injury In Mice And Its Mechanism

Part1Neuroprotective effect of Scriptaid, a new histone deacetylase inhibitor, against traumatic brain injury in miceTraumatic brain injury (TBI) is growing in prevalence among young adults with no effective therapeutic agent available to date. Despite much research effort, the complicated prognosis of this disorder has made the identification and development of a viable therapeutic agent elusive. However, recent reports indicate that histone deacetylase inhibitors (HDIs) can protect against tissue loss and functional deficits in TBI models. The epigenetic influence of HDIs on gene expression make them a novel class of pharmacological agents that may ameliorate disease conditions and enhance neuronal plasticity and survival. In this study, we characterized the protective capacity and mechanism of a new inhibitor of class I and II histone deacetylase, Scriptaid, against TBI.TBI was induced in C57/BL6mice by a controlled cortical impact (CCI) mouse model. Scriptaid was administered at doses between1.5to5.5mg/kg at0.5or12hr after injury. Short and long term motor deficits were determined using hang wire, cylinder and foot fault tests. Cognitive performance was detected in the Morris water maze tests29-34days post-TBI. Cresyl violet (CV) staining was used to assess the size of cortical lesion and calculate survival neurons at CA3. Post-traumatic neurodegeneration was analyzed using the MAP2immunolabeling method, combined with the Fluoro-jade method, which stains degenerating neuronal cell bodies. To ascertain the protective mechanism of Scriptaid, we looked for the activation of various protective pathways by immunohistochemical staining and Western blot analysis.Scriptaid elicited a dose-dependent decrease in lesion size at1.5to5.5mg/kg and a concomitant attenuation in motor and cognitive deficits when delivered30minutes post-injury in a model of moderate TBI. Comparable protection was achieved even when treatment was delayed to12hours post-injury. Furthermore, the protection of motor and cognitive functions was long lasting, as similar improvements were detected35days post-injury. The efficacy of Scriptaid was manifested as an increase in surviving neurons as well as the number/length of their processes within the CA3region of the hippocampus and the pericontusional cortex. Consistent with other HDIs, Scriptaid treatment prevented the decrease in phospho-AKT (p-AKT) and p-PTEN (phosphorylated phosphatase and tensin homolog deleted on chromosome10) induced by TBI in cortical and CA3hippocampal neurons. Furthermore, Scriptaid significantly increases ROCK1mRNA expression, which can regulate the phosphorylation, degradation, stability, and activity of PTEN. Notably, the p-AKT inhibitor LY294002attenuated the impact of Scriptaid, providing mechanistic evidence that Scriptaid functions partly by modulating the pro-survival AKT signaling pathway.In conclusion, the novel HDI Scriptaid effectively elicited short and long term protection against motor and cognitive deficits, both hall marks of TBI. Notably, Scriptaid prevented the TBI-induced loss in p-AKT and p-TEN levels whereas inhibition of AKT activation attenuated its protective effect. These findings make Scriptaid a promising new candidate for long term clinical protection against TBI. Part2Histone deacetylase inhibition prevents white matter lesions by titrating microglia/macrophage polarization following experimental traumatic brain injuryTraumatic brain injury (TBI) is a major cause of death and disability in young adults. TBI results not only in grey matter damage but also severe white matter injury (WMI). This WMI correlates with the long-term deficits in motor and cognitive function in TBI patients. Thus, the singular emphasis of most preclinical TBI studies on the protection of gray matter may explain the failure of experimental therapeutic agents in patients. We have studied the potent neuroprotective effects of Scriptaid, a novel inhibitor of class I/II histone deacetylase, against TBI. However, the effects of Scriptaid on long-term neurological functions and WMI following TBI remain elusive.TBI was induced in C57/BL6mice by a controlled cortical impact (CCI). RT-PCR and immunofluorescence staining for Ml and M2markers were performed to characterize phenotypic changes of microglia/macrophages in both grey and white matter. Scriptaid was administered at3.5mg/kg6hours after injury. Animals were sacrificed up to35days post-TBI. WMI was evaluated by myelin-specific Luxol fast blue staining, immunohistochemical staining for myelin basic protein (MBP) and neurofilament SMI-32. Electron microscopy was used to detect myelinated and unmyelinated fiber damage in the corpus callosum. White matter inflammation after TBI was characterized by the quantification of pro-inflammatory cytokines. Reverse-transcriptase polymerase chain reaction and immunohistochemical staining for M1and M2markers were performed to characterize microglia/macrophage phenotypic changes in cerebral white matter. In vitro experiments using a conditioned medium transfer system were used to further elucidate the effect of Scriptaid on microglia/macrophage polarization and the effect of microglia/macrophage phenotype on the fate of injured oligodendrocytes.Scriptaid elicited robust white matter protection at35days after TBI, as demonstrated by reductions in abnormally dephosphorylated neurofilament proteins SMI-32, increases in MBP staining and Iuxol blue positive myelin as well as preserved myelination. Scriptaid also mitigated inflammatory responses elicited by CCI. Moreover, microglia/macrophages surrounding the brain lesions were shifted from the M1phenotype to M2by Scriptaid. In vitro experiments revealed that Scriptaid primes microglial polarization towards the protective M2phenotype. As expected, Ml microglia-conditioned media exacerbated oxygen glucose deprivation (OGD)-induced oligodendrocytes death. In contrast, M2microglia-conditioned media protected oligodendrocytes against OGD.Our results indicate that HDAC inhibitor Scriptaid improves long term neurological outcomes after TBI through potent protection of white matter. Furthermore, HDAC inhibition may attenuate WMI by shifting the balance towards beneficial microglia/macrophage responses.