No abstract

4. The amplitudes of monosynaptically evoked IPSCs (elicited in the presence of 10,uM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50/M 2-amino-5-phosphonovaleric acid (APV)) were dramatically reduced during the DSI period. Weak stimulation produced small IPSCs and occasional 'failures' of transmission during the control period. The percentage of failures increased markedly during the DSI period. Moderate-intensity stimulation produced larger IPSCs that were often composed of distinguishable multiquantal components. All-ornone failures of multiquantal IPSC components also occurred during DSI. 5. The degree of paired-pulse IPSC depression did not change during DSI, whereas it was decreased, as expected, by baclofen. 6. We conclude that the data represent novel evidence that DSI is mediated by a retrograde signalling process possibly involving presynaptic axonal conduction block.

Mitochondria require cholesterol for biogenesis and membrane maintenance, and for the synthesis of steroids, oxysterols and hepatic bile acids. Multiple pathways mediate the transport of cholesterol from different subcellular pools to mitochondria. In steroidogenic cells, the steroidogenic acute regulatory protein (StAR) interacts with a mitochondrial protein complex to mediate cholesterol delivery to the inner mitochondrial membrane for conversion to pregnenolone. In non-steroidogenic cells, several members of a protein family defined by the presence of a StAR-related lipid transfer (START) domain play key roles in the delivery of cholesterol to mitochondrial membranes. Subdomains of the endoplasmic reticulum (ER), termed mitochondria-associated ER membranes (MAM), form membrane contact sites with mitochondria and may contribute to the transport of ER cholesterol to mitochondria, either independently or in conjunction with lipid-transfer proteins. Model systems of mitochondria enriched with cholesterol in vitro and mitochondria isolated from cells with (patho)physiological mitochondrial cholesterol accumulation clearly demonstrate that mitochondrial cholesterol levels affect mitochondrial function. Increased mitochondrial cholesterol levels have been observed in several diseases, including cancer, ischemia, steatohepatitis and neurodegenerative diseases, and influence disease pathology. Hence, a deeper understanding of the mechanisms maintaining mitochondrial cholesterol homeostasis may reveal additional targets for therapeutic intervention. Here we give a brief overview of mitochondrial cholesterol import in steroidogenic cells, and then focus on cholesterol trafficking pathways that deliver cholesterol to mitochondrial membranes in non-steroidogenic cells. We also briefly discuss the consequences of increased mitochondrial cholesterol levels on mitochondrial function and their potential role in disease pathology.

Evidence of male-to-female sexual transmission of Zika virus (ZIKV) and viral RNA in semen and sperm months after infection supports a potential role for testicular cells in ZIKV propagation. Here, we demonstrate that germ cells (GCs) are most susceptible to ZIKV. We found that only GCs infected by ZIKV, but not those infected by dengue virus and yellow fever virus, produce high levels of infectious virus. This observation coincides with decreased expression of interferon-stimulated gene Ifi44l in ZIKV-infected GCs, and overexpression of Ifi44l results in reduced ZIKV production. Using primary human testicular tissue, we demonstrate that human GCs are also permissive for ZIKV infection and production. Finally, we identified berberine chloride as a potent inhibitor of ZIKV infection in both murine and human testes. Together, these studies identify a potential cellular source for propagation of ZIKV in testes and a candidate drug for preventing sexual transmission of ZIKV.

The TTX-sensitive Ca(2+) current [I(Ca(TTX))] observed in cardiac myocytes under Na(+)-free conditions was investigated using patch-clamp and Ca(2+)-imaging methods. Cs(+) and Ca(2+) were found to contribute to I(Ca(TTX)), but TEA(+) and N-methyl-D-glucamine (NMDG(+)) did not. HEK-293 cells transfected with cardiac Na(+) channels exhibited a current that resembled I(Ca(TTX)) in cardiac myocytes with regard to voltage dependence, inactivation kinetics, and ion selectivity, suggesting that the cardiac Na(+) channel itself gives rise to I(Ca(TTX)). Furthermore, repeated activation of I(Ca(TTX)) led to a 60% increase in intracellular Ca(2+) concentration, confirming Ca(2+) entry through this current. Ba(2+) permeation of I(Ca(TTX)), reported by others, did not occur in rat myocytes or in HEK-293 cells expressing cardiac Na(+) channels under our experimental conditions. The report of block of I(Ca(TTX)) in guinea pig heart by mibefradil (10 microM) was supported in transfected HEK-293 cells, but Na(+) current was also blocked (half-block at 0.45 microM). We conclude that I(Ca(TTX)) reflects current through cardiac Na(+) channels in Na(+)-free (or "null") conditions. We suggest that the current be renamed I(Na(null)) to more accurately reflect the molecular identity of the channel and the conditions needed for its activation. The relationship between I(Na(null)) and Ca(2+) flux through slip-mode conductance of cardiac Na(+) channels is discussed in the context of ion channel biophysics and "permeation plasticity."

The neurotrophin receptor p75NTR has been implicated in mediating neuronal apoptosis after injury to the CNS. Despite its frequent induction in pathologic states, there is limited understanding of the mechanisms that regulate p75 NTR expression after injury. Here, we show that after focal cerebral ischemia in vivo or oxygen-glucose deprivation in organotypic hippocampal slices or neurons, p75 NTR is rapidly induced. A concomitant induction of proNGF, a ligand for p75 NTR , is also observed. Induction of this ligand/receptor system is pathologically relevant, as a decrease in apoptosis, after oxygen-glucose deprivation, is observed in hippocampal neurons or slices after delivery of function-blocking antibodies to p75 NTR or proNGF and in p75 NTR and ngf haploinsufficient slices. Furthermore, a significant decrease in infarct volume was noted in p75 NTR Ϫ/Ϫ mice compared with the wild type. We also investigated the regulatory mechanisms that lead to post-ischemic induction of p75 NTR . We demonstrate that induction of p75 NTR after ischemic injury is independent of transcription but requires active translation. Basal levels of p75 NTR in neurons are maintained in part by the expression of microRNA miR-592, and an inverse correlation is seen between miR-592 and p75 NTR levels in the adult brain. After cerebral ischemia, miR-592 levels fall, with a corresponding increase in p75 NTR levels. Importantly, overexpression of miR-592 in neurons decreases the level of ischemic injury-induced p75 NTR and attenuates activation of pro-apoptotic signaling and cell death. These results identify miR-592 as a key regulator of p75 NTR expression and point to a potential therapeutic candidate to limit neuronal apoptosis after ischemic injury.

No abstract

Peroxynitrite is usually considered as a neurotoxic nitric oxidederivative. However, an increasing body of evidence suggests that, at low concentrations, peroxynitrite affords transient cytoprotection, both in vitro and in vivo. Here, we addressed the signaling mechanism responsible for this effect, and found that rat cortical neurons in primary culture acutely exposed to peroxynitrite (0.1 mmol/L) rapidly elicited Akt-Ser 473 phos- Peroxynitrite (PN), the product of the reaction between nitric oxide (NO) and superoxide (O 2•) ) (Blough and Zafiriou 1985), is spontaneously formed in mammalian cells under physiological conditions (Radi et al. 2001;Possel et al. 2002). Initial work addressing the pathophysiology of PN yielded the widely accepted notion that this compound would be the long-term cytotoxic NO-derivative (Beckman et al. 1990;Radi et al. 1991). Peroxynitrite involvement in cytotoxicity is documented by detecting protein 3-nitrotyrosination, i.e. the 'footprinting' like evidence for in situ PN formation (Ischiropoulos et al. 1992;Greenacre and Ischiropoulos 2001;Beal 2002), associated with neurodegeneration and other disorders (Bolaños et al. 1997;Lipton 1999;Guix et al. 2005). However, in apparent contradiction with Received November 8, 2006; revised manuscript received January 5, 2007; accepted January 9, 2007. Address correspondence and reprint requests to Prof. Juan P. Bolaños, Departamento de Bioquímica y Biología Molecular, Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Salamanca, Spain. E-mail: jbolanos@usal.esAbbreviations used: 7-AAD, 7-amino-actinomycin D; Akt, protein kinase B; APC, allophycocyanin; BDNF, brain-derived neurotrophic factor; CAT, catalase; DAPI, 4¢-6-diamidino-2-phenylindole; GSH, glutathione ethyl ester; GFP, green fluorescent protein; Luc, luciferase; RNAi, RNA interference; PI3K, phosphoinositide-3-kinase; PFK-1, 6-phosphofructo-1-kinase; PN, peroxynitrite; PTP, protein tyrosine phosphatase; PTEN, phosphatase with tensin homology; shRNA, small hairpin RNA; SIN-1, 3-morpholinosydnonimine; SOD, superoxide dismutase; Trk, neurotrophin tyrosine kinase receptor.