Pharmacological inhibition of lipid peroxidation attenuates calpain-mediated cytoskeletal degradation after traumatic brain injury

Free radical-induced lipid peroxidation (LP) is critical in the evolution of secondary injury following traumatic brain injury (TBI). Previous studies in our laboratory demonstrated that U-83836E, a potent LP inhibitor, can reduce post-TBI LP along with an improved maintenance of mouse cortical mitochondrial bioenergetics and calcium (Ca(2+)) buffering following severe (1.0 mm; 3.5 m/s) controlled cortical impact TBI (CCI-TBI). Based upon this preservation of a major Ca(2+) homeostatic mechanism, we have now performed dose-response and therapeutic window analyses of the ability of U-83836E to reduce post-traumatic calpain-mediated cytoskeletal (α-spectrin) proteolysis in ipsilateral cortical homogenates at its 24 h post-TBI peak. In the dose-response analysis, mice were treated with a single i.v. dose of vehicle or U-83836E (0.1, 0.3, 1.3, 3.0, 10.0 or 30.0 mg/kg) at 15 min after injury. U-83836E produced a dose-related attenuation of α-spectrin degradation with the maximal decrease being achieved at 3.0 mg/kg. Next, the therapeutic window was tested by delaying the single 3 mg/kg i.v. dose from 15 min post-injury out to 1, 3, 6 or 12 h. No reduction in α-spectrin degradation was observed when the treatment delay was 1 h or longer. However, in a third experiment, we re-examined the window with repeated U-83836E dosing (3.0 mg/kg i.v. followed by 10 mg/kg i.p. maintenance doses at 1 and 3 h after the initial i.v. dose) which significantly reduced 24 h α-α-spectrin degradation even when treatment initiation was withheld until 12 h post-TBI. These results demonstrate the relationship between post-TBI LP, disruptions in neuronal Ca(2+) homeostasis and calpain-mediated cytoskeletal damage.     

Neuroprotection by quercetin via mitochondrial function adaptation in traumatic brain injury: PGC‐1α pathway as a potential mechanism

The aim of this study was to investigate the neuroprotective effects of quercetin in mouse models of traumatic brain injury (TBI) and the potential role of the PGC‐1α pathway in putative neuroprotection. Wild‐type mice were randomly assigned to four groups: the sham group, the TBI group, the TBI+vehicle group and the TBI+quercetin group. Quercetin, a dietary flavonoid used as a food supplement, significantly reduced TBI‐induced neuronal apoptosis and ameliorated mitochondrial lesions. It significantly accelerated the translocation of PGC‐1α protein from the cytoplasm to the nucleus. In addition, quercetin restored the level of cytochrome c, malondialdehyde and superoxide dismutase in mitochondria. Therefore, quercetin administration can potentially attenuate brain injury in a TBI model by increasing the activities of mitochondrial biogenesis via the mediation of the PGC‐1α pathway.     

Accumulation of non-erythroid alpha II-spectrin and calpain-cleaved alpha II-spectrin breakdown products in cerebrospinal fluid after traumatic brain injury in rats

Although a number of increased CSF proteins have been correlated with brain damage and outcome after traumatic brain injury (TBI), a major limitation of currently tested biomarkers is a lack of specificity for defining neuropathological cascades. Identification of surrogate biomarkers that are elevated in CSF in response to brain injury and that offer insight into one or more pathological neurochemical events will provide critical information for appropriate administration of therapeutic compounds for treatment of TBI patients. Non-erythroid alpha II-spectrin is a cytoskeletal protein that is a substrate of both calpain and caspase-3 cysteine proteases. As we have previously demonstrated, cleavage of alpha II-spectrin by calpain and caspase-3 results in accumulation of protease-specific spectrin breakdown products (SBDPs) that can be used to monitor the magnitude and temporal duration of protease activation. However, accumulation of alpha II-spectrin and alpha II-SBDPs in CSF after TBI has never been examined. Following a moderate level (2.0 mm) of controlled cortical impact TBI in rodents, native alpha II-spectrin protein was decreased in brain tissue and increased in CSF from 24 h to 72 h after injury. In addition, calpain-specific SBDPs were observed to increase in both brain and CSF after injury. Increases in the calpain-specific 145 kDa SBDP in CSF were 244%, 530% and 665% of sham-injured control animals at 24 h, 48 h and 72 h after TBI, respectively. The caspase-3-specific SBDP was observed to increase in CSF in some animals but to a lesser degree. Importantly, levels of these proteins were undetectable in CSF of uninjured control rats. These results indicate that detection of alpha II-spectrin and alpha II-SBDPs is a powerful discriminator of outcome and protease activation after TBI. In accord with our previous studies, results also indicate that calpain may be a more important effector of cell death after moderate TBI than caspase-3.     

Liraglutide is neurotrophic and neuroprotective in neuronal cultures and mitigates mild traumatic brain injury in mice

Traumatic brain injury (TBI), a brain dysfunction for which there is no present effective treatment, is often caused by a concussive impact to the head and affects an estimated 1.7 million Americans annually. Our laboratory previously demonstrated that exendin‐4, a long‐lasting glucagon‐like peptide 1 receptor (GLP‐1R) agonist, has neuroprotective effects in cellular and animal models of TBI. Here, we demonstrate neurotrophic and neuroprotective effects of a different GLP‐1R agonist, liraglutide, in neuronal cultures and a mouse model of mild TBI (mTBI). Liraglutide promoted dose‐dependent proliferation in SH‐SY5Y cells and in a GLP‐1R over‐expressing cell line at reduced concentrations. Pre‐treatment with liraglutide rescued neuronal cells from oxidative stress‐ and glutamate excitotoxicity‐induced cell death. Liraglutide produced neurotrophic and neuroprotective effects similar to those of exendin‐4 in vitro. The cAMP/PKA/pCREB pathway appears to play an important role in this neuroprotective activity of liraglutide. Furthermore, our findings in cell culture were well‐translated in a weight drop mTBI mouse model. Post‐treatment with a clinically relevant dose of liraglutide for 7 days in mice ameliorated memory impairments caused by mTBI when evaluated 7 and 30 days post trauma. These data cross‐validate former studies of exendin‐4 and suggest that liraglutide holds therapeutic potential for the treatment of mTBI.

     

Cytoplasmic gamma‐tubulin complex from brain contains nonerythroid spectrin

The newer member of the tubulin superfamily, γ‐tubulin, is known to mediate microtubule nucleation from the centrosome of eukaryotic cells with the aid of some other proteins. The major amount of γ‐tubulin is believed to be located in the centrosome before the onset of mitotic division. However, a considerable amount has been found in the cytoplasm in the form of a complex whose function is not well known. Microtubules are most abundant in brain tissues and brain microtubules have been extensively used in many in vitro studies. Thus, it is relevant to use brain tissue to characterize cytoplasmic γ‐tubulin complex. Here we show that cytoplasmic γ‐tubulin in brain tissues exists as a ring complex as in other tissues. Interestingly, along with the common members of the γ‐TuRC reported from several tissues and species, the purified brain cytoplasmic complex contains some high molecular weight proteins including α and β nonerythroid spectrin which are not found in other tissues. Immunohistochemical studies of brain tissue sections also show the co‐localization of γ‐tubulin and spectrin. The possible implications have been discussed. J. Cell. Biochem. 110: 1334–1341, 2010. © 2010 Wiley‐Liss, Inc.     

Dl‐3n‐butylphthalide improves traumatic brain injury recovery via inhibiting autophagy‐induced blood‐brain barrier disruption and cell apoptosis

Blood‐brain barrier (BBB) disruption and neuronal apoptosis are important pathophysiological processes after traumatic brain injury (TBI). In clinical stroke, Dl‐3n‐butylphthalide (Dl‐NBP) has a neuroprotective effect with anti‐inflammatory, anti‐oxidative, anti‐apoptotic and mitochondrion‐protective functions. However, the effect and molecular mechanism of Dl‐NBP for TBI need to be further investigated. Here, we had used an animal model of TBI and SH‐SY5Y/human brain microvascular endothelial cells to explore it. We found that Dl‐NBP administration exerts a neuroprotective effect in TBI/OGD and BBB disorder, which up‐regulates the expression of tight junction proteins and promotes neuronal survival via inhibiting mitochondrial apoptosis. The expressions of autophagy‐related proteins, including ATG7, Beclin1 and LC3II, were significantly increased after TBI/OGD, and which were reversed by Dl‐NBP treatment both in vivo and in vitro. Moreover, rapamycin treatment had abolished the effect of Dl‐NBP for TBI recovery. Collectively, our current studies indicate that Dl‐NBP treatment improved locomotor functional recovery after TBI by inhibiting the activation of autophagy and consequently blocking the junction protein loss and neuronal apoptosis. Dl‐NBP, as an anti‐inflammatory and anti‐oxidative drug, may act as an effective strategy for TBI recovery.     

TNF‐α–induced p53 activation induces apoptosis in neurological injury

It was previously confirmed that the apoptotic and necrotic neurons are found during the acute post‐traumatic period, suggesting the induction of apoptosis after traumatic brain injury (TBI). To further explore the involvement of apoptotic factors in TBI, an apoptosis antibody array was conducted to measure the alterations of apoptotic factors in rat brain cortex after TBI. As a result, the Neurological Severity Scale (NSS) scores after TBI were increased, and the cell morphology of the brain cortex was destructed with increased neuronal apoptosis. Furthermore, the caspase‐3 activity was increased, and the apoptotic‐related factors TNF‐α and p53 were up‐regulated in the brain cortex. More importantly, in vitro experiments demonstrated that down‐regulation of TNF‐α in oxygen‐glucose deprivation/reoxygenation (OGD/R) cells increased cell viability and decreased apoptosis and the p53 expression. These results suggested the involvement of TNF‐α–induced apoptotic signalling pathway by activating p53 in the molecular mechanism of neurological injury.     

Role of mitochondrial calcium uniporter‐mediated Ca2+ and iron accumulation in traumatic brain injury

Previous studies have suggested that the cellular Ca²⁺ and iron homeostasis, which can be regulated by mitochondrial calcium uniporter (MCU), is associated with oxidative stress, apoptosis and many neurological diseases. However, little is known about the role of MCU‐mediated Ca²⁺ and iron accumulation in traumatic brain injury (TBI). Under physiological conditions, MCU can be inhibited by ruthenium red (RR) and activated by spermine (Sper). In the present study, we used RR and Sper to reveal the role of MCU in mouse and neuron TBI models. Our results suggested that the Ca²⁺ and iron concentrations were obviously increased after TBI. In addition, TBI models showed a significant generation of reactive oxygen species (ROS), decrease in adenosine triphosphate (ATP), deformation of mitochondria, up‐regulation of deoxyribonucleic acid (DNA) damage and increase in apoptosis. Blockage of MCU by RR prevented Ca²⁺ and iron accumulation, abated the level of oxidative stress, improved the energy supply, stabilized mitochondria, reduced DNA damage and decreased apoptosis both in vivo and in vitro. Interestingly, Sper did not increase cellular Ca²⁺ and iron concentrations, but suppressed the Ca²⁺ and iron accumulation to benefit the mice in vivo. However, Sper had no significant impact on TBI in vitro. Taken together, our data demonstrated for the first time that blockage of MCU‐mediated Ca²⁺ and iron accumulation was essential for TBI. These findings indicated that MCU could be a novel therapeutic target for treating TBI.     

Phosphodiesterase isoform‐specific expression induced by traumatic brain injury

Traumatic brain injury (TBI) results in significant inflammation which contributes to the evolving pathology. Previously, we have demonstrated that cyclic AMP (cAMP), a molecule involved in inflammation, is down‐regulated after TBI. To determine the mechanism by which cAMP is down‐regulated after TBI, we determined whether TBI induces changes in phosphodiesterase (PDE) expression. Adult male Sprague Dawley rats received moderate parasagittal fluid‐percussion brain injury (FPI) or sham injury, and the ipsilateral, parietal cortex was analyzed by western blotting. In the ipsilateral parietal cortex, expression of PDE1A, PDE4B2, and PDE4D2, significantly increased from 30 min to 24 h post‐injury. PDE10A significantly increased at 6 and 24 h after TBI. Phosphorylation of PDE4A significantly increased from 6 h to 7 days post‐injury. In contrast, PDE1B, PD4A5, and PDE4A8 significantly decreased after TBI. No changes were observed with PDE1C, PDE3A, PDE4B1/3, PDE4B4, PDE4D3, PDE4D4, PDE8A, or PDE8B. Co‐localization studies showed that PDE1A, PDE4B2, and phospho‐PDE4A were neuronally expressed, whereas PDE4D2 was expressed in neither neurons nor glia. These findings suggest that therapies to reduce inflammation after TBI could be facilitated with targeted therapies, in particular for PDE1A, PDE4B2, PDE4D2, or PDE10A.     

Netrin‐1 alleviates subarachnoid haemorrhage‐induced brain injury via the PPARγ/NF‐KB signalling pathway

Netrin‐1 (NTN‐1) is a novel drug to alleviate early brain injury following subarachnoid haemorrhage (SAH). However the molecular mechanism of NTN‐1‐mediated protection against early brain injury following SAH remains largely elusive. This study aims to evaluate the effects and mechanisms of NTN‐1 in protecting SAH‐induced early brain injury. The endovascular perforation SAH model was constructed using male C57BL/6J mice, and recombinant NTN‐1 was administrated intravenously. Mortality rates, SAH grade, brain water content, neurological score and neuronal apoptosis were evaluated. The expression of PPARγ, Bcl‐2, Bax and nuclear factor‐kappa B (NF‐κB) were detected by Western blot. Small interfering RNA specific to NTN‐1 receptor, UNC5B, and a selective PPARγ antagonist, bisphenol A diglycidyl ether (BADGE), were applied in combination with NTN‐1. The results suggested that NTN‐1 improved the neurological deficits, reduced the brain water content and alleviated neuronal apoptosis. In addition, NTN‐1 enhanced PPARγ and Bcl‐2 expression and decreased the levels of Bax and NF‐κB. However, the neuroprotection of NTN‐1 was abolished by UNC5B and BADGE. In conclusion, our results demonstrated that NTN‐1 attenuates early brain injury following SAH via the UNC5B PPARγ/NF‐κB signalling pathway.     

Neuroprotective role of γ‐enolase in microglia in a mouse model of Alzheimer's disease is regulated by cathepsin X

γ‐Enolase is a neurotrophic‐like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which cleaves the C‐terminal end of the molecule. We have investigated the expression and colocalization of γ‐enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of γ‐enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C‐terminally cleaved form of γ‐enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of γ‐enolase in microglial cells in response to amyloid‐β peptide (Aβ) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with γ‐enolase proved to be neuroprotective against Aβ toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of γ‐enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid‐β‐related neurodegeneration.     

Up‐regulation and activation of the P2Y2 nucleotide receptor mediate neurite extension in IL‐1β‐treated mouse primary cortical neurons

The pro‐inflammatory cytokine interleukin‐1β (IL‐1β), whose levels are elevated in the brain in Alzheimer's and other neurodegenerative diseases, has been shown to have both detrimental and beneficial effects on disease progression. In this article, we demonstrate that incubation of mouse primary cortical neurons (mPCNs) with IL‐1β increases the expression of the P2Y₂ nucleotide receptor (P2Y₂R) and that activation of the up‐regulated receptor with UTP, a relatively selective agonist of the P2Y₂R, increases neurite outgrowth. Consistent with the accepted role of cofilin in the regulation of neurite extension, results indicate that incubation of IL‐1β‐treated mPCNs with UTP increases the phosphorylation of cofilin, a response absent in PCNs isolated from P2Y₂R^?/? mice. Other findings indicate that function‐blocking anti‐α_vβ_3/5 integrin antibodies prevent UTP‐induced cofilin activation in IL‐1β‐treated mPCNs, suggesting that established P2Y₂R/α_vβ_3/5 interactions that promote G₁₂‐dependent Rho activation lead to cofilin phosphorylation involved in neurite extension. Cofilin phosphorylation induced by UTP in IL‐1β‐treated mPCNs is also decreased by inhibitors of Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII), suggesting a role for P2Y₂R‐mediated and G_q‐dependent calcium mobilization in neurite outgrowth. Taken together, these studies indicate that up‐regulation of P2Y₂Rs in mPCNs under pro‐inflammatory conditions can promote cofilin‐dependent neurite outgrowth, a neuroprotective response that may be a novel pharmacological target in the treatment of neurodegenerative diseases.