Microglial activation and the nitric oxide/cGMP/PKG pathway underlie enhanced neuronal vulnerability to mitochondrial dysfunction in experimental multiple sclerosis.

During multiple sclerosis (MS), a close link has been demonstrated to occur between inflammation and neuro-axonal degeneration, leading to the hypothesis that immune mechanisms may promote neurodegeneration, leading to irreversible disease progression. Energy deficits and inflammation-driven mitochondrial dysfunction seem to be involved in this process. In this work we investigated, by the use of striatal electrophysiological field-potential recordings, if the inflammatory process associated with experimental autoimmune encephalomyelitis (EAE) is able to influence neuronal vulnerability to the blockade of mitochondrial complex IV, a crucial component for mitochondrial activity responsible of about 90% of total cellular oxygen consumption. We showed that during the acute relapsing phase of EAE, neuronal susceptibility to mitochondrial complex IV inhibition is markedly enhanced. This detrimental effect was counteracted by the pharmacological inhibition of microglia, of nitric oxide (NO) synthesis and its intracellular pathway (involving soluble guanylyl cyclase, sGC, and protein kinase G, PKG). The obtained results suggest that mitochondrial complex IV exerts an important role in maintaining neuronal energetic homeostasis during EAE. The pathological processes associated with experimental MS, and in particular the activation of microglia and of the NO pathway, lead to an increased neuronal vulnerability to mitochondrial complex IV inhibition, representing promising pharmacological targets.

Novel 3'-Processing Integrase Activity Assay by Real-Time PCR for Screening and Identification of HIV-1 Integrase Inhibitors.

The 3'-end processing (3'P) of each viral long terminal repeat (LTR) during human immunodeficiency virus type-1 (HIV-1) integration is a vital step in the HIV life cycle. Blocking the 3'P using 3'P inhibitor has recently become an attractive strategy for HIV-1 therapeutic intervention. Recently, we have developed a novel real-time PCR based assay for the detection of 3'P activity in vitro. The methodology usually involves biotinylated HIV-1 LTR, HIV-1 integrase (IN), and specific primers and probe. In this novel assay, we designed the HIV-1 LTR substrate based on a sequence with a homology to HIV-1 LTR labeled at its 3' end with biotin on the sense strand. Two nucleotides at the 3' end were subsequently removed by IN activity. Only two nucleotides labeled biotin were captured on an avidin-coated tube; therefore, inhibiting the binding of primers and probe results in late signals in the real-time PCR. This novel assay has successfully detected both the 3'P activity of HIV-1 IN and the anti-IN activity by Raltegravir and sodium azide agent. This real-time PCR assay has been shown to be effective and inexpensive for a high-throughput screening of novel IN inhibitors.