Deciphering the evolution of composite-type GSKIP in mitochondria and Wnt signaling pathways.

We previously revealed the origin of mammalian simple-type glycogen synthase kinase interaction protein (GSKIP), which served as a scavenger and a competitor in the Wnt signaling pathway during evolution. In this study, we investigated the conserved and nonconserved regions of the composite-type GSKIP by utilizing bioinformatics tools, site-directed mutagenesis, and yeast two-hybrid methods. The regions were denoted as the pre-GSK3ß binding site, which is located at the front of GSK3ß-binding sites. Our data demonstrated that clustered mitochondria protein 1 (CLU1), a type of composite-type GSKIP that exists in the mitochondria of all eukaryotic organisms, possesses the protein known as domain of unknown function 727 (DUF727), with a pre-GSK3ß-binding site and a mutant GSK3ß-binding flanking region. Another type of composite-type GSKIP, armadillo repeat containing 4 (ARMC4), which is known for cilium movement in vertebrates, contains an unintegrated DUF727 flanking region with a pre-GSK3ß-binding site (115SPxF118) only. In addition, the sequence of the GSK3ß-binding site in CLU1 revealed that Q126L and V130L were not conserved, differing from the ideal GSK3ß-binding sequence of simple-type GSKIP. We further illustrated two exceptions, namely 70 kilodalton heat shock proteins (Hsp70/DnaK) and Mitofilin in nematodes, that presented an unexpected ideal GSK3ß-binding region with a pre-GSK3ß sequence; this composite-type GSKIP could only occur in vertebrate species. Furthermore, we revealed the importance of the pre-GSK3ß-binding site (118F or 118Y) and various mutant GSK3ß-binding sites of composite-type GSKIP. Collectively, our data suggest that the new composite-type GSKIP starts with a DUF727 domain followed by a pre-GSK3ß-binding site, with the subsequent addition of the GSK3ß-binding site, which plays vital roles for CLU1, Mitofilin, and ARMC4 in mitochondria and Wnt signaling pathways during evolution.

Adenoviral-mediated glial cell line-derived neurotrophic factor gene transfer has a protective effect on sciatic nerve following constriction-induced spinal cord injury.

Neuropathic pain due to peripheral nerve injury may be associated with abnormal central nerve activity. Glial cell-line-derived neurotrophic factor (GDNF) can help attenuate neuropathic pain in different animal models of nerve injury. However, whether GDNF can ameliorate neuropathic pain in the spinal cord dorsal horn (SCDH) in constriction-induced peripheral nerve injury remains unknown. We investigated the therapeutic effects of adenoviral-mediated GDNF on neuropathic pain behaviors, microglial activation, pro-inflammatory cytokine expression and programmed cell death in a chronic constriction injury (CCI) nerve injury animal model. In this study, neuropathic pain was produced by CCI on the ipsilateral SCDH. Mechanical allodynia was examined with von Frey filaments and thermal sensitivity was tested using a plantar test apparatus post-operatively. Target proteins GDNF-1, GDNFRa-1, MMP2, MMP9, p38, phospho-p38, ED1, IL6, IL1ß, AIF, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, cleaved PARP, SPECTRIN, cleaved SPECTRIN, Beclin-1, PKCσ, PKCγ, iNOS, eNOS and nNOS were detected. Microglial activity was measured by observing changes in immunoreactivity with OX-42. NeuN and TUNEL staining were used to reveal whether apoptosis was attenuated by GDNF. Results showed that administrating GDNF began to attenuate both allodynia and thermal hyperalgesia at day 7. CCI-rats were found to have lower GDNF and GDNFRa-1 expression compared to controls, and GDNF re-activated their expression. Also, GDNF significantly down-regulated CCI-induced protein expression except for MMP2, eNOS and nNOS, indicating that the protective action of GDNF might be associated with anti-inflammation and prohibition of microglia activation. Immunocytochemistry staining showed that GDNF reduced CCI-induced neuronal apoptosis. In sum, GDNF enhanced the neurotrophic effect by inhibiting microglia activation and cytokine production via p38 and PKC signaling. GDNF could be a good therapeutic tool to attenuate programmed cell death, including apoptosis and autophagy, consequent to CCI-induced peripheral nerve injury.

Intrathecal lidocaine pretreatment attenuates immediate neuropathic pain by modulating Nav1.3 expression and decreasing spinal microglial activation.

BACKGROUND: Intrathecal lidocaine reverses tactile allodynia after nerve injury, but whether neuropathic pain is attenuated by intrathecal lidocaine pretreatment is uncertain. METHODS: Sixty six adult male Sprague-Dawley rats were divided into three treatment groups: (1) sham (Group S), which underwent removal of the L6 transverse process; (2) ligated (Group L), which underwent left L5 spinal nerve ligation (SNL); and (3) pretreated (Group P), which underwent L5 SNL and was pretreated with intrathecal 2% lidocaine (50 µl). Neuropathic pain was assessed based on behavioral responses to thermal and mechanical stimuli. Expression of sodium channels (Nav1.3 and Nav1.8) in injured dorsal root ganglia and microglial proliferation/activation in the spinal cord were measured on post-operative days 3 (POD3) and 7 (POD7). RESULTS: Group L presented abnormal behavioral responses indicative of mechanical allodynia and thermal hyperalgesia, exhibited up-regulation of Nav1.3 and down-regulation of Nav1.8, and showed increased microglial activation. Compared with ligation only, pretreatment with intrathecal lidocaine before nerve injury (Group P), as measured on POD3, palliated both mechanical allodynia (p < 0.01) and thermal hyperalgesia (p < 0.001), attenuated Nav1.3 up-regulation (p = 0.003), and mitigated spinal microglial activation (p = 0.026) by inhibiting phosphorylation (activation) of p38 MAP kinase (p = 0.034). p38 activation was also suppressed on POD7 (p = 0.002). CONCLUSIONS: Intrathecal lidocaine prior to SNL blunts the response to noxious stimuli by attenuating Nav1.3 up-regulation and suppressing activation of spinal microglia. Although its effects are limited to 3 days, intrathecal lidocaine pretreatment can alleviate acute SNL-induced neuropathic pain.

Ultrastructural changes of neuronal mitochondria after transient and permanent cerebral ischemia.

BACKGROUND AND PURPOSE: Mitochondrial swelling is one of the most striking and initial ultrastructural changes after acute brain ischemia. The purpose of the present study was to examine the role of reperfusion of the cerebral cortex after transient focal cerebral ischemia on neuronal mitochondrial damage. METHODS: Male Sprague-Dawley rats (n=16) were subjected to either temporary or permanent occlusion of the middle cerebral artery and bilateral carotid arteries. Three experimental conditions were compared: group I, permanent ischemia (3, 5, and 24 hours); group II, transient ischemia (2, 24 hours of reperfusion); and sham surgery. Anesthetized rats were killed by cardiac perfusion, and brain tissue was removed ipsilaterally and contralaterally from the ischemic core section of the frontoparietal cortex. Fixed tissue was prepared for electron microscopic examination, and electron microscopic thin sections of random neurons were photographed. Perinuclear neuronal mitochondria were analyzed in a blinded manner for qualitative ultrastructural changes (compared with sham control) by 2 independent investigators using an objective grading system. RESULTS: Cortical neuronal mitochondria exposed to severe ischemic/reperfusion conditions demonstrated dramatic signs of injury in the form of condensation, increased matrix density, and deposits of electron-dense material followed by disintegration by 24 hours. In contrast, mitochondria exposed to an equivalent time of permanent ischemia demonstrated increasing loss of matrix density with pronounced swelling followed by retention of their shape by 24 hours. CONCLUSIONS: Neuronal mitochondria undergoing transient versus permanent ischemia exhibit significantly different patterns of injury. Structural damage to neuronal mitochondria of the neocortex occurs more acutely and to a greater extent during the reperfusion phase in comparison to ischemic conditions alone. Further research is in progress to delineate the role of oxygen free radical production in the observed mitochondrial damage during postischemic reoxygenation.