Tumor necrosis factor-α (TNF-α) augments AMPA-induced Purkinje neuron toxicity.

It is well recognized that exposure of neurons to excessive levels of the excitatory neurotransmitter glutamate, termed glutamate excitotoxicity, contributes to the damage and degeneration seen in many acute and chronic neurological diseases. However, it is becoming increasingly evident that inflammation also can play a role in certain neurodegenerative diseases and inflammatory mediators, such as tumor necrosis factor-α (TNF-α), may directly interact with excitotoxic processes. In a postnatal rat cerebellar slice model, we found that TNF-α exacerbated AMPA-induced excitotoxicity in Purkinje neurons in a dose-dependent manner beyond the toxicity caused by AMPA alone. It also was shown that combinations of TNF-α and AMPA increased the mean intracellular activity of calpains, calcium-activated cysteine proteases that are known to contribute to cell death in Purkinje neurons. Additionally, these combinations augmented colbalt influx, a marker for calcium entry that selectively occurs through calcium permeable AMPA receptors only. Pharmacologic blockade of calcium permeable AMPA receptors with a specific antagonist, 1-naphthyl acetyl spermine (NASPM), reversed the apparent increase in AMPA receptor calcium permeability caused by TNF-α as measured by cobalt influx; caused a reduction in the Purkinje neuron calpain activity; and reversed the enhanced neurodegeneration induced by the combination of TNF-α and AMPA. From these studies we concluded that TNF-α augmented AMPA-induced toxicity in Purkinje neurons by increasing intracellular calcium flux through calcium permeable AMPA receptors, and this increase in calcium was directly involved in enhanced activation of calpains and a greater percentage of Purkinje neuron loss.

Effects of Amphotericin B on the expression of neurotoxic and neurotrophic factors in cultured microglia.

Amphotericin B (AmB) is a polyene antibiotic and reported to have therapeutic effects on prion diseases, in which the microglial activation has been suggested to play important roles by proliferating and producing various factors such as nitric oxide, proinflammatory cytokines, and so on. However, the therapeutic mechanism of AmB on prion diseases remains elusive. In the present study, we investigated the effects of AmB on cellular functions of rat primary cultured microglia. We found that AmB, similarly as lipopolysaccharide (LPS), could activate microglia to produce nitric oxide via inducible nitric oxide synthase. Both AmB and LPS also induced mRNA expressions of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in microglia. AmB also changed the expression levels of neurotrophic factors mRNAs. AmB and LPS significantly down-regulated the level of ciliary neurotrophic factor mRNA. However, AmB, but not LPS, significantly up-regulated the level of glial cell-line derived neurotrophic factor mRNA in microglia. In addition, brain-derived neurotrophic factor mRNA expression level was tending upward by treatment with AmB, but not with LPS. Taken together, these results suggest that AmB regulates the microglial activation in different manner from LPS and that microglia may participate in the therapeutic effects of AmB on prion diseases by controlling the expression and production of such mediators.

gamma-Conotoxin-PnVIIA, a gamma-carboxyglutamate-containing peptide agonist of neuronal pacemaker cation currents.

A novel gamma-carboxyglutamate-containing peptide, designated gamma-conotoxin-PnVIIA, is described from the venom of the molluscivorous snail Conus pennaceus. gamma PnVIIA, triggers depolarization and firing of action potential bursts in the caudodorsal neurons of Lymnaea. This effect is due to activation or enhancement of a slow inward cation current that may underly endogenous bursting activity of these neurons. The amino acid sequence of gamma PnVIIA was determined as DCTSWFGRCTVNS gamma CCSNSCDQTYC gamma-LYAFOS (where gamma is gamma-carboxyglutamate, O is trans-4-hydroxyproline), thus gamma PnVIIA belongs to the six cysteine four loop structural family of conotoxins, and is most homologous to the previously described excitatory conotoxin-TxVIIA. Interestingly, TxVIIA did not induce action potentials in Lymnaea caudodorsal neurons. gamma PnVIIA is the prototype of a new class of gamma-conotoxins that will provide tools for the study of voltage-gated pacemaker channels, which underly bursting processes in excitable systems.