One mechanism leading to neurodegeneration during Alzheimer's disease (AD) is amyloid beta peptide (Abeta) neurotoxicity. Abeta elicits in cultured central nervous system neurons a biphasic response: a low-dose neurotrophic response and a high-dose neurotoxic response. Previously we reported that NF-kappaB is activated by low doses of Abeta only. Here we show that NF-kappaB activation leads to neuroprotection. In primary neurons we found that a pretreatment with 0.1 microM Abeta-(1-40) protects against neuronal death induced with 10 microM Abeta-(1-40). As a known neuroprotective agent we next analyzed the effect of tumor necrosis factor alpha (TNF-alpha). Maximal activation of NF-kappaB was found with 2 ng/ml TNF-alpha. Pretreatment with TNF-alpha protected cerebellar granule cells from cell death induced by 10 microM Abeta-(1-40). This protection is described by an inverted U-shaped dose response and is maximal with a NF-kappaB-activating dose. The molecular specificity of this protective effect was analyzed by specific blockade of NF-kappaB activation. Overexpression of a transdominant negative IkappaB-alpha blocks NF-kappaB activation and potentiates Abeta-mediated neuronal apoptosis. Our findings show that activation of NF-kappaB is the underlying mechanism of the neuroprotective effect of low-dose Abeta and TNF-alpha. In accordance with these in vitro data we find that nuclear NF-kappaB immunoreactivity around various plaque stages of AD patients is reduced in comparison to age-matched controls. Taken together these data suggest that pharmacological NF-kappaB activation may be a useful approach in the treatment of AD and related neurodegenerative disorders.

The mechanism by which signals such as those produced by glutamate are transferred to the nucleus may involve direct transport of an activated transcription factor to trigger long-term transcriptional changes.

Neonicotinoids represent a class of insect-selective ligands of nicotinic acetylcholine receptors. Imidacloprid, the first commercially used neonicotinoid insecticide, has been studied on neuronal preparations from many insects to date. Here we report first intrinsic binding data of thiamethoxam, using membranes from Myzus persicae Sulzer and Aphis craccivora Koch. In both aphids, specific binding of [3H]thiamethoxam was sensitive to temperature, while the absolute level of non-specific binding was not affected. In M persicae, binding capacity (Bmax) for [3H]thiamethoxam was ca 450 fmol mg(-1) of protein at 22 degrees C and ca 700 fmol mg(-1) of protein at 2 degrees C. The negative effect of increased temperature was reversible and hence not due to some destructive process. The affinity for [3H]thiamethoxam was less affected by temperature: Kd was ca 11 nM at 2 degrees C and ca 15 nM at 22 degrees C. The membranes also lost binding sites for [3H]thiamethoxam during prolonged storage at room temperature, and upon freezing and thawing. In A craccivora, [3H]thiamethoxam was bound with a capacity of ca 1000 fmol mg(-1) protein and an affinity of ca 90 nM, as measured at 2 degrees C. Overall, the in vitro temperature sensitivity of [3H]thiamethoxam binding was in obvious contrast to the behaviour of [3H]imidacloprid studied in parallel. Moreover, the binding of [3H]thiamethoxam was inhibited by imidacloprid in a non-competitive mode, as shown with M persicae. In our view, these differences demonstrate that thiamethoxam and imidacloprid, which represent different structural sub-classes of neonicotinoids, do not share the same binding site or mode. This holds also for other neonicotinoids, as we report in a companion article.

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The present study investigates the pharmacological profile of P2-purinoceptors modulating noradrenaline release from cultured chick sympathetic neurons. ATP (30 microM-3 mM) and 2-methylthio-ATP (3-100 microM), but not alpha, beta-methylene-ATP (up to 100 microM), caused a significant facilitation of electrically evoked [3H]-noradrenaline release when added 2 min before depolarization. The facilitation declined with time of exposure suggesting receptor desensitization. The facilitatory effect was markedly diminished by the P2-purinoceptor antagonists reactive blue 2 (3 microM) and suramin (300 microM), but not changed by mecamylamine (10 microM), a nicotinic receptor antagonist. At 1 mM and higher concentrations, ATP added for 12 min, inhibited noradrenaline release; release was virtually abolished by 6 mM ATP. The inhibitory effect of ATP was slightly diminished by suramin but not affected by reactive blue 2. Electrically evoked [3H]-noradrenaline release remained unaffected in the presence of the adenosine (P1)-receptor agonists R(-)N6-(2-phenylisopropyl)adenosine (R-PIA), 2-[p-(2-carboxyethyl) phenylethylamino]-5'-N-ethylcarboxamidoadenosine (NECA), and N6-2-(4-aminophenyl)ethyladenosine (APNEA), used up to 1 microM. The present results confirm the existence of two P2-purinoceptors affecting noradrenaline release: 1) a facilitatory receptor which is activated by 2-methylthio-ATP as well as ATP, and blocked by suramin as well as reactive blue 2, and 2) an inhibitory receptor which is activated by ATP, only slightly affected by suramin but not at all by reactive blue 2 and does not belong to the established P2-purinoceptor subtypes.

The ATP-induced increase in tritium outflow from cultured chick sympathetic neurons prelabelled with [3H]-noradrenaline was investigated. Seven days-old dissociated cell cultures of embryonic paravertebral ganglia, loaded with [3H]-noradrenaline (0.05 microM), were superfused in the presence of (+)-oxaprotiline and exposed to ATP, ATP-analogues, or 1,1-dimethyl-4-piperazinium (DMPP) for 2 min. ATP (3 microM-3 mM), 2-methylthio-ATP (3-100 microM), as well as DMPP (10 and 100 microM) induced a significant overflow of tritium. The EC50-value of ATP was 20 microM. Both the ATP-induced and the DMPP-induced tritium overflow was Ca(2+)-dependent and sensitive to tetrodotoxin (0.3 microM) and omega-conotoxin (0.1 microM); in addition, it was inhibited by the alpha 2-adrenoceptor agonist 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline (UK-14,304; 1 microM). The effects of ATP and DMPP were not additive. The ATP-induced as well as the DMPP-induced overflow of tritium was diminished by the P2-purinoceptor antagonists suramin (300 microM) and reactive blue 2 (3 microM); in all 4 cases, the inhibition amouted to approximately 40%. The tritium overflow induced by ATP or DMPP was almost abolished by the nicotinic receptor antagonist mecamylamine (10 microM) and markedly inhibited by hexamethonium (100 microM). Neither ATP nor electrical stimulation caused an overflow of tritium from cultures loaded with [3H]-choline. The results suggest that ATP at mumolar concentrations induces noradrenaline release from cultured chick sympathetic neurons via an action on a subclass of the nicotinic cholinoceptor.

The neonicotinoids are the most successful chemical class of insecticides reaching sales of more than 630 Mio $ in 2001, mainly due to the excellent market performance of imidacloprid and thiamethoxam. The insect nicotinic acetylcholine receptors (nAChRs) are the targets for these compounds, which are highly effective against a variety of sucking and chewing insects. Compared with the other neonicotinoid sales products, thiamethoxam binds in a different way, possibly to a different site of nAChRs in aphids. To gain further insight into the different modes of binding, a research program applying the photoaffinity labeling technique was started. A series of novel candidate photoaffinity probes containing a 5-azido-6-chloropyridin-3-ylmethyl group were prepared from 5-azido-6-chloropyridin-3-ylmethyl chloride, which was obtained in three steps from 6-chloropyridin-3-ylmethyl chloride. These probes showed good to excellent contact/feeding and systemic activity against Myzus persicae, however, they were at least 4- to 16-fold less effective against Aphis craccivora, Nilaparvata lugens, Spodoptera littoralis, and Diabrotica balteata than the neonicotinoid sales products. In general, the introduction of an azide group at C(5) of the 6-chloropyridin-3-ylmethyl substituent resulted in reduced potency as well as in a narrower pest spectrum. In competition binding assays with [3H]imidacloprid, analogues of imidacloprid, clothianidin, thiacloprid and thiamethoxam containing a 5-azido-6-chloropyridin-3-ylmethyl group showed high displacing potency with nAChRs from Aphis and Myzus (Ki values: 2 to 27 nM) suggesting that these compounds are valuable candidate photoaffinity probes. Taking into account the biological screening activity as well as the receptor binding potency, 1-(5-azido-6-chloropyridin-3-ylmethyl)-2-nitroimino-imidazolidine, N-(5-azido-6-chloropyridin-3-ylmethyl)-N?-methyl-N?-nitroguanidine and 3-(5-azido-6-chloropyridin-3-ylmethyl)-2-cyanoimino-thiazolidine were identified as the preferred candidate neonicotinoid photoaffinity probes to study the imidacloprid binding site.

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