RESUMO
Traumatic brain injury (TBI), even at mild levels, can activate matrix metalloproteinases (MMPs) and the induction of neuroinflammation that can result in blood brain barrier breakdown and neurodegeneration. MMP2 has a significant role in neuroinflammation and neurodegeneration by modulating the chemokine CXCL12α (stromal cell derived factor SDF-1α) signaling pathway and the induction of apoptosis. SDF-1α is responsible for cell proliferation and differentiation throughout the nervous system and is also implicated in various neurodegenerative illnesses. We hypothesized that TBI leads to MMP2 activation and cleavage of the N-terminal 4 amino acid residues of CXCL12α with generation of the highly neurotoxic fragment SDF-1(5-67). Using an in vitro stretch-injury model of rat neuronal cultures and the in vivo fluid percussion injury (FPI) model in rats, we found that oxidative stress has a significant role in the activation of MMP2. This is initiated by the induction of free radical generating enzyme NADPH oxidase 1 (NOX1). Induction of NOX1 correlated well with the signatures of oxidative stress marker, 4HNE in the injured neuronal cultures and cerebral cortex of rats. Further, using MMP2 siRNA and pharmacological MMP2 inhibitor, ARP100, we established the neurodegenerative role of MMP2 in cleaving SDF-1α to a neurotoxic fragment SDF-1(5-67). By immunofluorescence, western blotting and TUNEL experiments, we show the cleaved form of SDF leads to apoptotic cell death in neurons. This work identifies a new potential therapeutic target to reduce the complications of brain damage in TBI.
Assuntos
Lesões Encefálicas Traumáticas/enzimologia , Quimiocina CXCL12/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Degeneração Neural/enzimologia , Degeneração Neural/genética , Animais , Apoptose/efeitos dos fármacos , Lesões Encefálicas Traumáticas/genética , Caspase 3/biossíntese , Caspase 3/genética , Sobrevivência Celular/genética , Células Cultivadas , Quimiocina CXCL12/genética , Ativação Enzimática , Técnicas de Silenciamento de Genes , Metaloproteinase 2 da Matriz/genética , Inibidores de Metaloproteinases de Matriz/farmacologia , NADPH Oxidase 1/biossíntese , NADPH Oxidase 1/genética , Neurônios/efeitos dos fármacos , Estresse Oxidativo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
We investigated the hypothesis that high Ca2+ influx during traumatic brain injury induces the activation of the caspase-1 enzyme, which triggers neuroinflammation and cell apoptosis in a cell culture model of neuronal stretch injury and an in vivo model of fluid percussion injury (FPI). We first established that stretch injury causes a rapid increase in the intracellular Ca2+ level, which activates interleukin-converting enzyme caspase-1. The increase in the intracellular Ca2+ level and subsequent caspase-1 activation culminates into neuroinflammation via the maturation of IL-1ß. Further, we analyzed caspase-1-mediated apoptosis by TUNEL staining and PARP western blotting. The voltage-gated sodium channel blocker, tetrodotoxin, mitigated the stretch injury-induced neuroinflammation and subsequent apoptosis by blocking Ca2+ influx during the injury. The effect of tetrodotoxin was similar to the caspase-1 inhibitor, zYVAD-fmk, in neuronal culture. To validate the in vitro results, we demonstrated an increase in caspase-1 activity, neuroinflammation and neurodegeneration in fluid percussion-injured animals. Our data suggest that neuronal injury/traumatic brain injury (TBI) can induce a high influx of Ca2+ to the cells that cause neuroinflammation and cell death by activating caspase-1, IL-1ß, and intrinsic apoptotic pathways. We conclude that excess IL-1ß production and cell death may contribute to neuronal dysfunction and cognitive impairment associated with TBI.