Donepezil, rivastigmine, and galantamine are three drugs with acetylcholinesterase (AChE)-inhibiting activity that are currently being used to treat patients suffering from Alzheimer's disease. We have studied the neuroprotective effects of these drugs, in comparison with nicotine, on cell death caused by ␤-amyloid (A␤) and okadaic acid, two models that are relevant to Alzheimer's pathology, in the human neuroblastoma cell line SH-SY5Y. Galantamine and donepezil showed a U-shaped neuroprotective curve against okadaic acid toxicity; maximum protection was achieved at 0.3 M galantamine and at 1 M donepezil; at higher concentrations, protection was diminished. Rivastigmine showed a concentration-dependent effect; maximum protection was achieved at 3 M. When apoptosis was induced by A␤ [25][26][27][28][29][30][31][32][33][34][35] , galantamine, donepezil, and rivastigmine showed maximum protection at the same concentrations: 0.3, 1, and 3 M, respectively. Nicotine also afforded protection against A␤-and okadaic acid-induced toxicity. The neuroprotective effects of galantamine, donepezil, and nicotine were reversed by the ␣7 nicotinic antagonist methyllycaconitine but not by the ␣4␤2 nicotinic antagonist dihydro-␤-erythroidine. The phosphoinositide 3-kinase (PI3K)-Akt blocker 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) reversed the protective effects of galantamine, donepezil, and nicotine but not that of rivastigmine. In contrast, the bcl-2 antagonist ethyl[2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA 14-1) reversed the protective effects of the three AChE inhibitors and that of nicotine. Our results show that galantamine, donepezil, and rivastigmine afford neuroprotection through a mechanism that is likely unrelated to AChE inhibition. Such neuroprotection seemed to be linked to ␣7 nicotinic receptors and the PI3K-Akt pathway in the case of galantamine and donepezil but not for rivastigmine.Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common form of dementia in the elderly population. Clinically, patients with AD show progressive deterioration of all cognitive functions, resulting in their incapacitation. AD is characterized by the presence of two kinds of abnormal protein deposits, amyloid plaques and neurofibrillary tangles (NFTs) in specific areas of the brain, and finally by the atrophy of the affected brain regions, which results from extensive losses of synapses and neurons (Terry et al., 1981(Terry et al., , 1991Price et al., 1991;Arriagada et al., 1992). Amyloid plaques are extracellular deposits containing ␤-amyloid peptide (A␤) as the major core deposits. A␤ is a 39-to 43-amino acid peptide fragment derived through proteolysis from an integral membrane protein known as A␤ precursor protein. The basis for the ␤-amyloid hypothesis arises from various studies showing that A␤ is toxic to neurons; for example, there is increased A␤ release and apoptotic cell

(4). Calmodulin (CaM) seems to be involved in this process through two CaM binding sites located one at the C-terminal (5) and the other at the ankyrin repeat domain in the N-terminal end of the protein (6, 7). Deletion of these sites relieves TRPV1 desensitization and tachyphylaxis. Other factors that can modulate the activity of TRPV1 are changes of pH or temperature, inflammatory mediators, and phosphorylation (8). These modulators may be involved in sensitization to pain. A large part of TRPV1 naturally expressed in DRG neurons locates in endomembranes rather than in the plasma membrane (9 -12), and a similar situation has been reported for other TRP channels. The function of these endomembrane channels is not known, although it has been speculated that they may represent a reserve pool that could be rapidly mobilized to the plasma membrane when required (13). In this direction, it has been reported that activation of protein kinase C (14), nerve growth factor-induced phosphorylation via Src kinase (15) or coupling with phosphoinositide 3-kinase (16) promotes insertion of ER-located TRPV1 channels (TRPV1 ER ) into the plasma membrane (TRPV1 PM ). This mobilization could be the basis of inflammatory sensitization and hyperalgesia.On the other hand, previous work has shown that TRPV1 ER channels are functional (9 -12, 17) and that its activation leads to alterations of ER and mitochondria, followed by cell death (9, 18). Cell death due to ER stress following ER Ca 2ϩ emptying by TRPV1 ER stimulation has also been documented in human lung cells (19). Transfection of HEK293T cells with TRPV1 reproduces the neuronal model with expression of functional TRPV1 ER and TRPV1 PM channels (9,16,18,20,21).In all of the previous studies, the effects of TRPV1 on [Ca 2ϩ ] ER were inferred from the changes of the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] C ). We can now monitor directly [Ca 2ϩ ] ER in living cells using ER-targeted aequorins (22-24). Here we have studied in detail the release of Ca 2ϩ from ER induced by activation of TRPV1 in DRG neurons and in HEK293T cells expressing TRPV1 channels.

Genetically encoded calcium indicators allow monitoring subcellular Ca 2+ signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca 2+ sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg 2+ , tunable Ca 2+ affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca 2+ indicators for organelles and becomes a valuable tool for in vivo Ca 2+ imaging applications.calcium signalling | Golgi apparatus | hippocampus | motor neuron