Cerebellar granule cells undergo apoptosis in culture after deprivation of potassium and serum. During this process we found that tau, a neuronal microtubule-associated protein that plays a key role in the maintenance of neuronal architecture, and the pathology of which correlates with intellectual decline in Alzheimer's disease, is cleaved. The final product of this cleavage is a soluble dephosphorylated tau fragment of 17 kDa that is unable to associate with microtubules and accumulates in the perikarya of dying cells. The appearance of this 17 kDa fragment is inhibited by both caspase and calpain inhibitors, suggesting that tau is an in vivo substrate for both of these proteases during apoptosis. Tau cleavage is correlated with disruption of the microtubule network, and experiments with colchicine and taxol show that this is likely to be a cause and not a consequence of tau cleavage. These data indicate that tau cleavage and change in phosphorylation are important early factors in the failure of the microtubule network that occurs during neuronal apoptosis. Furthermore, this study introduces new insights into the mechanism(s) that generate the truncated forms of tau present in Alzheimer's disease.

The altered function and͞or structure of tau protein is postulated to cause cell death in tauopathies and Alzheimer's disease. However, the mechanisms by which tau induces neuronal death remain unclear. Here we show that overexpression of human tau and of some of its N-terminal fragments in primary neuronal cultures leads to an N-methyl-D-aspartate receptor (NMDAR)-mediated and caspase-independent cell death. Death signaling likely originates from stimulation of extrasynaptic NR2B-subunit-containing NMDARs because it is accompanied by dephosphorylation of cAMP-response-element-binding protein (CREB) and it is inhibited by ifenprodil. Interestingly, activation of NMDAR leads to a crucial, sustained, and delayed phosphorylation of extracellular-regulated kinases 1 and 2, whose inhibition largely prevents tau-induced neuronal death. Moreover, NMDAR involvement causes the fatal activation of calpain, which, in turn, degrades tau protein into a 17-kDa peptide and possibly other highly toxic N-terminal peptides. Some of these peptides are hypothesized, on the basis of our in vitro experiments, to initiate a negative loop, ultimately leading to cell death. Thus, inhibition of calpain largely prevents tau degradation and cell death. Our findings unravel a cellular mechanism linking tau toxicity to NMDAR activation and might be relevant to Alzheimer's disease and tauopathies where NMDARmediated toxicity is postulated to play a pivotal role. extracellular-regulated kinase ͉ mitogen-activated protein kinase ͉ neurodegenerative diseases ͉ glutamate receptors ͉ Alzheimer's disease

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The amyloid precursor protein (APP) and its proteolytic product amyloid beta (A␤) are associated with both familial and sporadic forms of Alzheimer disease (AD). Aberrant expression and function of microRNAs has been observed in AD. Here, we show that in rat hippocampal neurons cultured in vitro, the down-regulation of Argonaute-2, a key component of the RNA-induced silencing complex, produced an increase in APP levels. Using site-directed mutagenesis, a microRNA responsive element (RE) for miR-101 was identified in the 3-untranslated region (UTR) of APP. The inhibition of endogenous miR-101 increased APP levels, whereas lentiviral-mediated miR-101 overexpression significantly reduced APP and A␤ load in hippocampal neurons. In addition, miR-101 contributed to the regulation of APP in response to the proinflammatory cytokine interleukin-1␤ (IL-l␤). Thus, miR-101 is a negative regulator of APP expression and affects the accumulation of A␤, suggesting a possible role for miR-101 in neuropathological conditions. Alzheimer disease (AD)2 is the most common form of dementia in aged individuals and is characterized by A␤ plaques, which contain A␤ aggregates and neurofibrillary tangles which consist primarily of aggregated forms of the microtubule-stabilizing protein tau (reviewed in Ref. 1). A␤ peptides are derived from processing of the type I transmembrane protein APP through sequential cleavages by ␤ and ␥ secretase (2-3). The A␤ load during pathology leads to neurological dysfunction. APP is linked to AD; familial AD can be caused by increased expression of APP due to either genomic duplication (4 -5) or regulatory sequence alterations (6). Among the physiological and pathological activators of APP expression (7-8) is the proinflammatory cytokine IL-1␤ (9). IL-1␤ is produced in the central nervous system (CNS) in response to damage and influences neuronal function by interacting with the type I IL-1 receptor expressed on neurons (10 -11). IL-1␤ is overexpressed in AD (12) and has been implicated in initiation and progression of AD pathology (13). In addition, IL-1␤ promotes APP transcription (14) and translation (9) in various cell types. Transcriptional and post-transcriptional regulation of APP expression has been widely studied and correlated to AD pathogenesis (15-16). Both cell type-specific promoter elements (17) and regulatory elements in the 5Ј-and 3Ј-UTRs of APP mRNA have been identified (9, 18).MicroRNAs are an intriguing class of small noncoding RNA molecules which, in mammals, regulate gene expression primarily by imperfect base pairing with the

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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...

We investigated the potential role of the ubiquitin proteolytic system in the death of cerebellar granule neurons induced by reduction of extracellular potassium. Inhibitors of proteasomal function block apoptosis if administered at onset of this process, but they do not exert such effect when added 2-3 hr later. The same inhibitors also prevent caspase-3 activity and calpain-caspase-3-mediated processing of tau protein, suggesting that proteasomes are involved upstream of the caspase activation. Although the proteasomes seem to play an early primary role in programmed cell death, we found that with progression of apoptosis, during the execution phase, a perturbation in normal ubiquitin-proteasome function occurs, and high levels of ubiquitinated proteins accumulate in the cytoplasm of dying cells. Such accumulation correlates with a progressive decline of proteasome chymotrypsin and trypsinlike activities and, to a lower extent, of postacidic-like activity. Both intracytoplasmic accumulation of ubiquitinated proteins and decline of proteasome function are reversed by the pancaspase inhibitor Z-VAD-fmk. The decline in proteasome function is accompanied by, and likely attributable to, a marked and progressive decline of deubiquitinating activities. The finding that the proteasomes are early involved in apoptosis and that ubiquitinated proteins accumulate during this process prospect granule neurons as a model system aimed at correlating these events with neurodegenerative diseases.

Btg1 belongs to a family of cell cycle inhibitory genes. We observed that Btg1 is highly expressed in adult neurogenic niches, i.e., the dentate gyrus and subventricular zone (SVZ). Thus, we generated Btg1 knockout mice to analyze the role of Btg1 in the process of generation of adult new neurons. Ablation of Btg1 causes a transient increase of the proliferating dentate gyrus stem and progenitor cells at post-natal day 7; however, at 2 months of age the number of these proliferating cells, as well as of mature neurons, greatly decreases compared to wild-type controls. Remarkably, adult dentate gyrus stem and progenitor cells of Btg1-null mice exit the cell cycle after completing the S phase, express p53 and p21 at high levels and undergo apoptosis within 5 days. In the SVZ of adult (two-month-old) Btg1-null mice we observed an equivalent decrease, associated to apoptosis, of stem cells, neuroblasts, and neurons; furthermore, neurospheres derived from SVZ stem cells showed an age-dependent decrease of the self-renewal and expansion capacity. We conclude that ablation of Btg1 reduces the pool of dividing adult stem and progenitor cells in the dentate gyrus and SVZ by decreasing their proliferative capacity and inducing apoptosis, probably reflecting impairment of the control of the cell cycle transition from G1 to S phase. As a result, the ability of Btg1-null mice to discriminate among overlapping contextual memories was affected. Btg1 appears, therefore, to be required for maintaining adult stem and progenitor cells quiescence and self-renewal.