Deletion of mitochondrial anchoring protects dysmyelinating shiverer: implications for progressive MS.

The demyelinating disease multiple sclerosis (MS) has an early inflammatory phase followed by an incurable progressive phase with subdued inflammation and poorly understood neurodegenerative mechanism. In this study, we identified various parallelisms between progressive MS and the dysmyelinating mouse model Shiverer and then genetically deleted a major neuron-specific mitochondrial anchoring protein Syntaphilin (SNPH) from the mouse. Prevailing evidence suggests that deletion of SNPH is harmful in demyelination. Surprisingly, SNPH deletion produces striking benefits in the Shiverer by prolonging survival, reducing cerebellar damage, suppressing oxidative stress, and improving mitochondrial health. In contrast, SNPH deletion does not benefit clinical symptoms in experimental autoimmune encephalomyelitis (EAE), a model for early-phase MS. We propose that deleting mitochondrial anchoring is a novel, specific treatment for progressive MS.

Molecular mechanisms of apoptosis in cerebral ischemia: multiple neuroprotective opportunities.

Cerebral ischemia/reperfusion (I/R) injury triggers multiple and distinct but overlapping cell signaling pathways, which may lead to cell survival or cell damage. There is overwhelming evidence to suggest that besides necrosis, apoptosis do contributes significantly to the cell death subsequent to I/R injury. Both extrinsic and intrinsic apoptotic pathways play a vital role, and upon initiation, these pathways recruit downstream apoptotic molecules to execute cell death. Caspases and Bcl-2 family members appear to be crucial in regulating multiple apoptotic cell death pathways initiated during I/R. Similarly, inhibitor of apoptosis family of proteins (IAPs), mitogen-activated protein kinases, and newly identified apoptogenic molecules, like second mitochondrial-activated factor/direct IAP-binding protein with low pI (Smac/Diablo), omi/high-temperature requirement serine protease A2 (Omi/HtrA2), X-linked mammalian inhibitor of apoptosis protein-associated factor 1, and apoptosis-inducing factor, have emerged as potent regulators of cellular apoptotic/antiapoptotic machinery. All instances of cell survival/death mechanisms triggered during I/R are multifaceted and interlinked, which ultimately decide the fate of brain cells. Moreover, apoptotic cross-talk between major subcellular organelles suggests that therapeutic strategies should be optimally directed at multiple targets/mechanisms for better therapeutic outcome. Based on the current knowledge, this review briefly focuses I/R injury-induced multiple mechanisms of apoptosis, involving key apoptotic regulators and their emerging roles in orchestrating cell death programme. In addition, we have also highlighted the role of autophagy in modulating cell survival/death during cerebral ischemia. Furthermore, an attempt has been made to provide an encouraging outlook on emerging therapeutic approaches for cerebral ischemia.

Regulation of neural progenitor cell proliferation by D609: potential role for ERK.

Tricyclodecan-9-yl-xanthogenate (D609) has been shown to possess both neuroprotective and anti-proliferative properties. We investigated the role of D609 in reducing the proliferation of neural progenitor cells in vitro. D609 decreased the expression of cyclin D1 after 1 day but not 2 or 4 days in culture, indicating the possible degradation/inactivation of drug in the medium. Consistent with this notion, spectral analysis showed the maximum absorbance of D609 (100 µM) at 300 nm, which decreased by ~30 % following incubation at 37 °C for 24 h. Further experiments revealed that incubation of neural progenitor cells with D609 decreased the phosphorylation of extracellular signal-regulated kinase (ERK) but not Akt. In addition, increasing the concentration of B27 (1-4 %), but not FGF2, diminished the effect of D609 on cell proliferation. These results together suggest that D609 may curtail the proliferation of neural progenitor cells by decreasing the ERK-mediated expression of cyclin D1 and may have a therapeutic potential in containing the proliferation of tumor stem cells.

Anti-proliferative effects of tricyclodecan-9-yl-xanthogenate (D609) involve ceramide and cell cycle inhibition.

Tricyclodecan-9-yl-xanthogenate (D609) inhibits phosphatidylcholine (PC)-phospholipase C (PLC) and/or sphingomyelin (SM) synthase (SMS). Inhibiting SMS can increase ceramide levels, which can inhibit cell proliferation. Here, we examined how individual inflammatory and glia cell proliferation is altered by D609. Treatment with 100-µM D609 significantly attenuated the proliferation of RAW 264.7 macrophages, N9 and BV-2 microglia, and DITNC(1) astrocytes, without affecting cell viability. D609 significantly inhibited BrdU incorporation in BV-2 microglia and caused accumulation of cells in G(1) phase with decreased number of cells in the S phase. D609 treatment for 2 h significantly increased ceramide levels in BV-2 microglia, which, following a media change, returned to control levels 22 h later. This suggests that the effect of D609 may be mediated, at least in part, through ceramide increase via SMS inhibition. Western blots demonstrated that 2-h treatment of BV-2 microglia with D609 increased expression of the cyclin-dependent kinase (Cdk) inhibitor p21 and down-regulated phospho-retinoblastoma (Rb), both of which returned to basal levels 22 h after removal of D609. Exogenous C8-ceramide also inhibited BV-2 microglia proliferation without loss of viability and decreased BrdU incorporation, supporting the involvement of ceramide in D609-mediated cell cycle arrest. Our current data suggest that D609 may offer benefit after stroke (Adibhatla and Hatcher, Mol Neurobiol 41:206-217, 2010) through ceramide-mediated cell cycle arrest, thus restricting glial cell proliferation.

Endoplasmic reticulum stress plays critical role in brain damage after cerebral ischemia/reperfusion in rats.

The endoplasmic reticulum(ER) stress plays a vital role in mediating ischemic neuronal cell death. However, very little is known about the role of ER stress in mediating pathophysiological reactions to acute brain injuries. An attempt was therefore made to assess the role of cerebral ischemia/reperfusion (I/R) induced ER stress and its modulation on outcome of ischemic insult. Focal cerebral ischemia was induced in rats by middle cerebral artery occlusion (MCAO) for 2 h followed by varying time points of reperfusion. The brain loci specific and time-dependent alterations were seen in the expression pattern of molecular markers, i.e., heat-shock protein 70 (HSP70) for cytoplasmic dysfunction, glucose-regulated protein 78 (GRP78), Caspase-12, C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153 (CHOP/GADD153), activating transcription factor 4 (ATF-4), and Processed X-box protein 1 (xbp1) mRNA for ER dysfunction. Further, histological examinations indicated pronounced brain damage, massive neuronal loss, and DNA fragmentation predominantly in the striatum and cortex. The enhanced expression of GRP78, Caspase-12, CHOP/GADD153, ATF4 and processing of xbp1 mRNA in the affected brain regions clearly indicate the critical involvement of ER-mediated cell death/survival mechanisms and also collectively demonstrated the activation of unfolded protein response (UPR). Moreover, Salubrinal, a selective inhibitor of eIF2alpha dephosphorylation was used to counteract ER stress, which significantly increased the phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2alpha), leading to reduced brain damage after I/R injury. Therefore, inhibition of ER stress following I/R injury may be used as key therapeutic target for neuroprotection.