Background and Purpose— The Na + /Ca 2+ exchanger, by mediating Ca 2+ and Na + fluxes in a bidirectional way across the synaptic plasma membrane, may play a pivotal role in the events leading to anoxic damage. In the brain, there are 3 different genes coding for 3 different proteins: NCX1, NCX2, and NCX3. The aim of this study was to determine whether NCX1, NCX2, and NCX3 might play a differential role in the development of cerebral injury induced by permanent middle cerebral artery occlusion (pMCAO). Methods— By means of Western blotting, NCX1, NCX2, and NCX3 protein expression was evaluated in the ischemic core and in the remaining nonischemic area of the slice at different time intervals starting from ischemia induction. The role of each isoform was also assessed with antisense oligodeoxynucleotides (ODNs) targeted for each isoform. These ODNs were continuously intracerebroventricularly infused with an osmotic minipump (1 μL/h) for 48 hours, 24 hours before pMCAO. Results— The results showed that after pMCAO all 3 NCX proteins were downregulated in ischemic core; NCX3 decreased in periinfarctual area whereas NCX1 and NCX2 were unchanged. The ODNs for NCX1 and NCX3 gene products were capable of inducing an increase in the ischemic lesion and to worsen neurological scores. Conclusions— The results of this study suggest that in the neuroprotective effect exerted by NCX during ischemic injury, the major role is prevalently exerted by NCX1 and NCX3 gene products.
Here, we report that interruption of NGF or BDNF signaling in hippocampal neurons rapidly activates the amyloidogenic pathway and causes neuronal apoptotic death. These events are associated with an early intracellular accumulation of PS1 N-terminal catalytic subunits and of APP C-terminal fragments and a progressive accumulation of intra-and extracellular A aggregates partly released into the culture medium. The released pool of A induces an increase of APP and PS1 holoprotein levels, creating a feedforward toxic loop that might also cause the death of healthy neurons. These events are mimicked by exogenously added A and are prevented by exposure to -and ␥-secretase inhibitors and by antibodies directed against A peptides. The same cultured neurons deprived of serum die, but APP and PS1 overexpression does not occur, A production is undetectable, and cell death is not inhibited by anti-A antibodies, suggesting that hippocampal amyloidogenesis is not a simple consequence of an apoptotic trigger but is due to interruption of neurotrophic signaling. Hippocampal neurons are among the most severely affected cells in AD (1). BDNF and NGF carry out a variety of actions on these neurons and are involved in the clinical and pathophysiological signs of AD (2, 3). In AD, a reduction in neurotrophin levels occurs in some areas of the CNS, and neurotrophic factors have begun to be used in clinical trials to prevent or to reduce neuronal cell loss (4) or to improve hippocampal neurogenesis in adult and aged male rats (5). In a previous study of NGF-differentiated PC12 cells, we reported a close correlation between NGF deprivation and activation of the amyloidogenic route (6), thus broadening to this clonal line the link between amyloidogenesis and cell death formerly reported in cultured cerebellar granule cells (7,8). Although this clonal cell line has contributed to elucidating a large number of neuronal properties, because of its clonal neoplastic origin, it might not provide information specifically connected to the pathological events actually occurring in the neurons of the adult brain. Therefore, to clarify the molecular events found in NGF-differentiated PC12 cells, we resorted to primary hippocampal neurons and also tried to assess whether another neurotrophin, such as BDNF, shares the same antiamyloidogenic activity previously found with NGF.In the present study, we confirm and largely extend findings obtained in NGF-differentiated PC12 cells demonstrating that interruption of the NGF (or BDNF) signal induces death through an intra-and extracellular accumulation of A aggregates and activation of a feed-forward toxic loop that also causes the death of healthy neurons. These events are associated with the formation of varicosities along neurites and with an accumulation of APP C-terminal fragments in neurons undergoing death. Furthermore, A peptides released in medium induce an increase of APP and PS1 holoprotein levels, and all these events are prevented by ␥-and -secretase inhibitors or by antibody direct...
Nerve growth factor (NGF) exerts a trophic, antiapoptotic action on several neuronal targets, including the clonal cell line PC12. In the current study, we demonstrate that withdrawal of this neurotrophin from PC12 differentiated cells causes overproduction of amyloid-β (Aβ) peptides, which are the most toxic protein fragments directly implicated in the development of Alzheimer disease (AD), concomitantly with cell death by apoptosis. Aβ production and apoptotic death, occurring after withdrawal from NGF-differentiated PC12 cells, are completely inhibited by βand γ-secretase inhibitors and by antibodies directed against Aβ peptides, favouring maintenance of PC12 morphology and neuritic network. These peptides are partially released and largely deposited as aggregates only soluble with strong detergent treatment generally employed to dissolve senile plaques. Furthermore, partial silencing of APP mRNA, by siRNA, reduces not only the extent of Aβ production but also apoptotic death. Aβ production and apoptosis are also induced in differentiated PC12 cells by kinase inhibitors of Trk-A, the high affinity receptor of NGF and, in this case, the co-incubation with βand γ-secretase inhibitors totally revert apoptosis.
Alzheimer disease (AD) is a human neurodegenerative disease characterized by co-existence of extracellular senile plaques (SP) and neurofibrillary tangles (NFT) associated with an extensive neuronal loss, primarily in the cerebral cortex and hippocampus. Several studies suggest that caspase(s)-mediated neuronal death occurs in cellular and animal AD models as well as in human brains of affected patients, although an etiologic role of apoptosis in such neurodegenerative disorder is still debated. This review summarizes the experimental evidences corroborating the possible involvement of apoptosis in AD pathogenesis and discusses the usefulness of ad hoc devised in vitro approaches to study how caspase(s), amyloidogenic processing and tau metabolism might reciprocally interact leading to neuronal death.
The aim of this study was to correlate dopamine receptors and D(2) isoform expression with the cabergoline effect on alpha-subunit secretion in vitro and tumor mass in vivo in clinically nonfunctioning pituitary tumors. Eighteen patients were subjected to neurosurgery, and a tumor sample was used for dopamine receptor and D(2) isoform expression evaluation by RT-PCR and the in vitro functional studies. After neurosurgery, nine of 18 patients with persistent tumor were treated with cabergoline and tumor mass was evaluated before and after 1 yr treatment. D(2) receptor was expressed in 67% of cases. D(2long) was found in 50%, D(2short) in 17%, and both D(2) isoforms in 33% of cases. D(4) receptor was also expressed in 17% of cases. The in vitro inhibition of alpha-subunit concentration was found in 56% of cases and was associated with D(2) expression (chi(2) = 5.6; P < 0.05). After 1 yr of cabergoline treatment, tumor shrinkage was evident in 56% of patients and was associated with D(2) expression (chi(2) = 5.6; P < 0.05). The expression of D(2short) rather than D(2long) isoform is associated with the most favorable response of the tumor to cabergoline treatment. In conclusion, this study demonstrates D(2) receptor expression and function in nearly 70% of cases, suggesting a role of this drug in the treatment schedule of clinically nonfunctioning pituitary tumors.
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