Identification of a caspase-derived N-terminal tau fragment in cellular and animal Alzheimer's disease models.

Biochemical modifications of tau proteins have been proposed to be among the earliest neurobiological changes in Alzheimer's disease (AD) and correlate better with cognitive symptoms than do beta-amyloid plaques. We have recently reported that adenovirus-mediated overexpression of the NH2 26-230aa tau fragment evokes a potent NMDA-mediated neurotoxic effect in primary neuronal cultures. In order to assess whether such N-terminal tau fragment(s) are indeed produced during apoptosis or neurodegeneration in vivo, we attempted to ascertain their presence in cell and animal models using an anti-tau antibody directed against the N-terminal sequence of human protein located downstream of the caspase(s)-cleavage site DRKD(25)-QGGYTMHQDQ. We provide biochemical evidence that a caspase(s)-cleaved NH2-terminal tau fragment of 20-22 kDa, consistent with the size of the NH2 26-230aa neurotoxic fragment of tau, is generated in vitro in differentiated human SH-SY5Y cells undergoing apoptosis by BDNF withdrawal or following treatment with staurosporine. In addition this NH2-terminally cleaved tau fragment, whose expression correlates with a significant up-regulation of caspase(s) activity, is also specifically detected in vivo in the hippocampus of 15 month-old AD11 transgenic mice, a model in which a progressive AD-like neurodegeneration is induced by the expression of transgenic anti-NGF antibodies. The results support the idea that aberrant activation of caspase(s), following apoptotic stimuli or neurodegeneration insults, may produce one or more toxic NH2 tau fragments, that further contribute to propagate and increase cellular dysfunctions in AD.

Substance P provides neuroprotection in cerebellar granule cells through Akt and MAPK/Erk activation: evidence for the involvement of the delayed rectifier potassium current.

In the current study, we have evaluated the ability of substance P (SP) and other neurokinin 1 receptor (NK1) agonists to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death (S-K5). We also established the presence of SP high affinity NK1 transcripts and the NK1 protein localization in the membrane of a sub-population of CGCs. Moreover, SP significantly and dose-dependently reduced the Akt 1/2 and Erk1/2 dephosphorylation induced by S-K5 conditions, as demonstrated by Western blot analysis. Surprisingly, in SP-treated CGCs caspase-3 activity was not inhibited, while the calpain-1 activity was moderately reduced. Corroborating this result, SP blocked calpain-mediated cleavage of tau protein, as demonstrated by the reduced appearance of a diagnostic fragment of 17 kDa by Western blot analysis. In addition, SP induced a significant reduction of the delayed rectifier K+ currents (Ik) in about 42% of the patched neurons, when these were evoked with depolarizing potential steps. Taken together, the present results demonstrate that the activation of NK1 receptors expressed in CGCs promote the neuronal survival via pathways involving Akt and Erk activation and by inhibition of Ik which can contribute to the neuroprotective effect of the peptide.

Interplay of the E box, the cyclic AMP response element, and HTF4/HEB in transcriptional regulation of the neurospecific, neurotrophin-inducible vgf gene.

vgf is a neurotrophin response-specific, developmentally regulated gene that codes for a neurosecretory polypeptide. Its transcription in neuronal cells is selectively activated by the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3, which induce survival and differentiation, and not by epidermal growth factor. We studied a short region of the rat vgf promoter which is essential for its regulated expression. A cyclic AMP response element (CRE) within this region is necessary for NGF induction of vgf transcription. Two sites upstream of CRE, an E box and a CCAAT sequence, bind nuclear protein complexes and are involved in transcriptional control. The E box has a dual role. It acts as an inhibitor in NIH 3T3 fibroblasts, together with a second E box located downstream, and as a stimulator in the NGF-responsive cell line PC12. By expression screening, we have isolated the cDNA for a basic helix-loop-helix transcription factor, a homolog of the HTF4/HEB E protein, that specifically binds the vgf promoter E box. The E protein was present in various cell lines, including PC12 cells, and was a component of a multiprotein nuclear complex that binds the promoter in vitro. The E box and CRE cooperate in binding to this complex, which may be an important determinant for neural cell-specific expression.

Proteasome involvement and accumulation of ubiquitinated proteins in cerebellar granule neurons undergoing apoptosis.

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 trypsin-like 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 pan-caspase 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.

Role of N-terminal tau domain integrity on the survival of cerebellar granule neurons.

Although the role of the microtubule-binding domain of the tau protein in the modulation of microtubule assembly is widely established, other possible functions of this protein have been poorly investigated. We have analyzed the effect of adenovirally mediated expression of two fragments of the N-terminal portion - free of microtubule-binding domain - of the tau protein in cerebellar granule neurons (CGNs). We found that while the expression of the tau (1-230) fragment, as well as of full-length tau, inhibits the onset of apoptosis, the tau (1-44) fragment exerts a powerful toxic action on the same neurons. The antiapoptotic action of tau (1-230) is exerted at the level of Akt-mediated activation of the caspase cascade. On the other hand, the toxic action of the (1-44) fragment is not prevented by inhibitors of CGN apoptosis, but is fully inhibited by NMDA receptor antagonists. These findings point to a novel, physiological role of the N-terminal domain of tau, but also underlay that its possible proteolytic truncation mediated by apoptotic proteases may generate a highly toxic fragment that could contribute to neuronal death.

Expression in murine and human neuroblastoma cell lines of VGF, a tissue specific protein.

Screening by different means has demonstrated the presence, in human and murine neuroblastoma cell lines, of VGF, a gene product identified in a limited number of neuronal and endocrine cells. Indirect immunofluorescence and Western and Northern blot analyses have shown the presence of this protein in some of the tested lines, confirming that VGF is not an ubiquitous molecule. Further studies, using human SK-N-BE and murine N18TG2 lines, showed that VGF expression is upregulated during differentiation, suggesting that various species, including man, express VGF and regulate it in a similar manner. The subcellular localization of the protein, which is associated with vesicles, its electrophoretic molecular profile and its specific release under different conditions are all consistent with results reported in other cells. Neuroblastomas are thus added to the class of VGF-positive cells and provide a new in vitro model for investigation of the structural and functional properties of this protein.

Does the term 'trophic' actually mean anti-amyloidogenic? The case of NGF.

The term trophic is widely used to indicate a general pro-survival action exerted on target cells by different classes of extracellular messengers, including neurotrophins (NTs), a family of low-molecular-weight proteins whose archetypal member is the nerve growth factor (NGF). The pro-survival action exerted by NTs results from a coordinated activation of multiple metabolic pathways, some of which have only recently come to light. NGF has been shown to exert a number of different, experimentally distinguishable effects on neurons, such as survival, differentiation of target neurons, growth of nerve fibers and their guidance (tropism) toward the source of its production. We have proposed a more complete definition of the NGF trophic action that should also include its newly discovered property of inhibiting the amyloidogenic processing of amyloid precursor protein (APP), which is among the first hypothesized primary trigger of Alzheimer's disease (AD) pathogenesis. This inhibitory action appears to be mediated by a complex series of molecular events and by interactions among NGF receptors (TrkA and p75), APP processing and tau metabolic fate and function.

Characterization of virulence factors in the newly described Salmonella enterica serotype Keurmassar emerging in Senegal (sub-Saharan Africa).

From 2000 to 2001, nine strains of Salmonella enterica belonging to the new serotype Keurmassar have been isolated from human and poultry samples at the Senegalese National Salmonella and Shigella Reference Laboratory at the Pasteur Institute, in Dakar. All strains carried virulence factors including Salmonella Pathogenicity Islands (SPI)-1, -2, -3 and -5 encoded genes. Strains did not harbour virulence plasmid. Ribotyping analysis revealed a single clone identical to Salmonella Decatur isolated in Zimbabwe. These data suggest that strains are closely related, and may have been spread clonally. In this new serotype, insertion sequence IS200 is not present.

Molecular cloning and characterization of the human VGF promoter region.

The VGF gene encodes a secretory protein that is expressed in a cell type-restricted pattern in neuroendocrine cells and is up-regulated by nerve growth factor (NGF) in the rat pheochromocytoma PC12 cell line. Here we report the isolation and characterization of the 5'-terminal region of the human VGF gene. In addition to a TATA box and a CCAAT box located at canonical distances from the transcription start site, the human VGF promoter contains several consensus sequences for different transcription factors, including a cyclic AMP response element and an AP-1 element, several GC boxes, and sequences homologous to other neuronal promoters. Transient transfection analysis demonstrates that 2.3 kb of the 5'-flanking sequence acts as a tissue-specific promoter, efficiently used only by neuronal cells that express endogenous VGF. Deletion analysis reveals that a positive regulatory region is located between nucleotides -458 to -204. Negative cis-acting elements that repress promoter activity in cell lines that do not normally express VGF are located between nucleotides -2,305 and -573 and between -458 and -204. The 5'-flanking region of the human VGF gene confers responsiveness to NGF, cyclic AMP, and phorbol ester treatment.

Cloning, structural organization analysis, and chromosomal assignment of the human gene for the neurosecretory protein VGF.

The Vgf gene was originally identified as a 2.7-kb cDNA fragment isolated from nerve growth factor-treated PC12 cells by differential display against PC12 cells. It is transcribed solely in subpopulations of neuroendocrine cells in vivo and it is induced by neurotrophins in target cells in vitro. The single-copy human VGF gene was isolated from a genomic library. The gene spans approximately 6 kb and contains two exons. The entire VGF protein is encoded by exon 2, while exon 1 contains only 5'-untranslated sequence. The structural organization of the human gene is similar to that described for the rat Vgf gene (S. R. J. Salton et al., 1991, Mol. Cell. Biol. 11: 2335-2349) and both the translated and the untranslated regions show a high degree of sequence homology to the rat gene. Northern blot analysis revealed a single transcript of approximately 2.7 kb that was detected only in mRNA preparations from brain. The gene was assigned to chromosome 7q22 by fluorescence in situ hybridization.

NMDA receptor modulation by a conditioned medium derived from rat cerebellar granule cells.

Our previous studies have shown that the response to the excitotoxic action of glutamate by cultured cerebellar granule cells depends upon the cell density or the volume of medium in which they have been grown: the higher the cell density or the lower the volume, the higher the response to glutamate. We have hypothesized that this variable response is due to the formation in culture of a glutamate-sensitizing activity GSA more abundantly in conditioned medium derived from high-density or low-volume cultures than that present in low-density or high volume cultures and capable of restoring sensitivity in previously resistant granule cells. In order to elucidate the mechanism of action of glutamate-sensitizing activity, we measured the extent and function of NMDA receptors in low- and high-volume cultures and assessed the effect of glutamate-sensitizing activity on the same receptors. We found that under high-volume conditions the extent of MK-801 binding, the amount of NMDA receptor type 1, the currents evoked in whole cells after an NMDA pulse and the response of cultured cells to this ligand were markedly reduced compared with low-volume cultures. Addition of glutamate-sensitizing activity to high-volume cultures increased their glutamate sensitivity, the NMDA-evoked currents, the extent of MK-801 binding and the amount of NMDA receptor type 1 protein present. The corresponding mRNA transcripts, on the contrary, were unchanged in high-volume, low-volume and high-volume GSA-treated cultures.

Tissue-specific processing of the neuroendocrine protein VGF.

VGF is a neuroendocrine-specific gene product that is up-regulated by nerve growth factor in the PC12 cell line. In rat neuroendocrine tissues two polypeptides of 90 and 80 kDa were detected by an antiserum to an N-terminal domain of VGF (from residues 4 to 240). In parallel, an antiserum directed against the C-terminal nonapeptide of VGF (from residues 609 to 617) revealed several additional posttranslational products. Peptides of apparent molecular sizes of 20, 18, and 10 kDa were prominent in nerve tissues and the hypophysis but absent in the adrenal medulla, and their relative abundance varied in distinct regions of the CNS. In PC12 cells VGF was proteolytically processed only after nerve growth factor treatment, and primary cultures of rat cerebellar granule cells accumulated the low-molecular-weight forms of VGF during in vitro maturation. In these cells the specific cleavages of VGF occurred in a postendoplasmic reticulum compartment; the processed forms were enriched in the secretory vesicles and were preferentially secreted upon cell membrane depolarization. Distinct differential distribution in the CNS and in vitro release of such posttranslational products indicate that these species may represent biologically relevant forms of VGF that play a role in neuronal communication.

Characterization of focal liver lesions in real time using harmonic imaging with high mechanical index and contrast agent levovist.

OBJECTIVE: The aim of this study was to characterize focal hepatic lesions using agent detection imaging and Levovist. MATERIALS AND METHODS: Sixty-five patients (21 male and 44 female; age range, 8-82 years; mean +/- standard deviation, 58.1 +/- 14.5 years) were independently evaluated by two observers in a blinded manner using stored sonographic images. Seventy-five lesions were found: 15 hepatocellular carcinomas, nine focal nodular hyperplasias, two adenomas, 21 hemangiomas, 23 metastases, and five regenerative nodules. Nine patients were excluded (six because of technical failures, three with unproven diagnoses). New high-mechanical-index software was used to reveal power harmonic responses from contrast microbubble destruction. After a venous bolus injection of 4 g of Levovist at a strength of 400 mg/mL, delayed imaging was used to study lesion enhancement in the arterial, portal, and parenchymal phases. Two comparisons were made. The first was between the B-mode image and the first contrast-enhanced image after the flash. The second was between color Doppler sonograms and real-time contrast-enhanced perfusion images. RESULTS: Contrast-enhanced images after the flash and real-time contrast-enhanced images revealed more information for the characterization of the lesion than did gray-scale and color Doppler images (p < 0.0001, Wilcoxon's signed rank test). Different types of lesions showed statistically significant differences in enhancement during each of the three vascular phases (p < 0.005, Kruskal-Wallis test). Lesions with lower contrast enhancement were metastases and regenerating nodules. Good agreement was present between the two observers; differences were not statistically significant (p > 0.05). CONCLUSION: Agent detection imaging with Levovist increased diagnostic confidence in the characterization of focal hepatic lesions as compared with standard sonography.

Isolation and characterization of VGF peptides in rat brain. Role of PC1/3 and PC2 in the maturation of VGF precursor.

The neurotrophin responsive gene vgf is widely expressed in central and peripheral neurones, and in certain neuroendocrine cell populations. Its encoded VGF precursor protein (proVGF1: 617 amino acids in rat, 615 in man, > 85% homology) gives rise to several low molecular weight species. We studied a range of neuroendocrine and neuronal cells, in which VGF-processing products were prominent with an apparent molecular weight of 20 and 10 kDa (VGF20 and VGF10, respectively). Such peptides were recognized by antibodies specific for the C-terminal rat VGF nonapeptide, thus indicating that they included the C-terminus of proVGF. Ectopic expression of the neuroendocrine-specific prohormone convertases PC1/3 or PC2 in GH3 cells showed that both could generate VGF20, while VGF10 was preferentially produced by PC1/3. Site-directed mutagenesis was used to identify the KRKRKK(488) motif as the target within VGF sequence which leads to the production of VGF20. Molecular characterization of rat VGF10, on the other hand, revealed that this peptide is produced by cleavage at the RPR(555) site. By the combined use of high-resolution separation techniques, matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectrometry and manual Edman degradation we identified in rat brain a VGF fragment analogous to bovine peptide V and two novel peptides also derived from the C-terminal region of proVGF.

Tau cleavage and dephosphorylation in cerebellar granule neurons undergoing apoptosis.

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.