Caspase-2 mediates neuronal cell death induced by beta-amyloid.

beta-amyloid (Abeta) has been proposed to play a role in the pathogenesis of Alzheimer's disease (AD). Deposits of insoluble Abeta are found in the brains of patients with AD and are one of the pathological hallmarks of the disease. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. In our current study, treatment with nitric oxide generators protected against Abeta-induced death, whereas inhibition of nitric oxide synthase afforded no protection, suggesting that formation of peroxynitrite is not critical for Abeta-mediated death. Previous studies have shown that aggregated Abeta can induce caspase-dependent apoptosis in cultured neurons. In all of the neuronal populations studied here (hippocampal neurons, sympathetic neurons, and PC12 cells), cell death was blocked by the broad spectrum caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone and more specifically by the downregulation of caspase-2 with antisense oligonucleotides. In contrast, downregulation of caspase-1 or caspase-3 did not block Abeta(1-42)-induced death. Neurons from caspase-2 null mice were totally resistant to Abeta(1-42) toxicity, confirming the importance of this caspase in Abeta-induced death. The results indicate that caspase-2 is necessary for Abeta(1-42)-induced apoptosis in vitro.

Abnormal microtubule packing in processes of SF9 cells expressing the FTDP-17 V337M tau mutation.

Mutations in the gene for the microtubule associated protein, tau have been identified for fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). In vitro data have shown that FTDP-17 mutant tau proteins have a reduced ability to bind microtubules and to promote microtubule assembly. Using the baculovirus system we have examined the effect of the V337M mutation on the organization of the microtubules at the ultrastructural level. Our results show that the organization of the microtubules is disrupted in the presence of V337M tau with greater distances between the microtubules and fewer microtubules per process.

AnkyrinG is associated with the postsynaptic membrane and the sarcoplasmic reticulum in the skeletal muscle fiber.

Ankyrins are a multi-gene family of peripheral proteins that link ion channels and cell adhesion molecules to the spectrin-based skeleton in specialized membrane domains. In the mammalian skeletal myofiber, ankyrins were immunolocalized in several membrane domains, namely the costameres, the postsynaptic membrane and the triads. Ank1 and Ank3 transcripts were previously detected in skeletal muscle by northern blot analysis. However, the ankyrin isoforms associated with these domains were not identified, with the exception of an unconventional Ank1 gene product that was recently localized at discrete sites of the sarcoplasmic reticulum. Here we study the expression and subcellular distribution of the Ank3 gene products, the ankyrinsG, in the rat skeletal muscle fiber. Northern blot analysis of rat skeletal muscle mRNAs using domain-specific Ank3 cDNA probes revealed two transcripts of 8.0 kb and 5.6 kb containing the spectrin-binding and C-terminal, but not the serine-rich, domains. Reverse transcriptase PCR analysis of rat skeletal muscle total RNA confirmed the presence of Ank3 transcripts that lacked the serine-rich and tail domains, a major insert of 7813 bp at the junction of the spectrin-binding and C-terminal domains that was previously identified in brain Ank3 transcripts. Immunoblot analysis of total skeletal muscle homogenates using ankyrinG-specific antibodies revealed one major 100 kDa ankyrinG polypeptide. Immunofluorescence labeling of rat diaphragm cryosections showed that ankyrin(s)G are selectively associated with (1) the depths of the postsynaptic membrane folds, where the voltage-dependent sodium channel and N-CAM accumulate, and (2) the sarcoplasmic reticulum, as confirmed by codistribution with the sarcoplasmic reticulum Ca2+-ATPase (SERCA 1). At variance with ankyrin(s)G, ankyrin(s)R (ank1 gene products) accumulate at the sarcolemma and at sarcoplasmic structures, in register with A-bands. Both ankyrin isoforms codistributed over Z-lines and at the postsynaptic membrane. These data extend the notion that ankyrins are differentially localized within myofibers, and point to a role of the ankyrinG family in the organization of the sarcoplasmic reticulum and the postsynaptic membrane.

Peripherin is tyrosine-phosphorylated at its carboxyl-terminal tyrosine.

Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf-9 cells and SW-13 cl. 2 vim- cells. The identity of peripherin as a tyrosine-phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW-13 cl. 2 vim- cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy-terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild-type peripherin and peripherin Y474F form a filamentous network in SW-13 cl. 2 vim- cells. This indicates that tyrosine phosphorylation of the peripherin C-terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.

Cell cycle-dependent phosphorylation of nucleoporins and nuclear pore membrane protein Gp210.

During mitosis in higher eukaryotic cells, the nuclear envelope membranes break down into distinct populations of vesicles and the proteins of the nuclear lamina and the nuclear pore complexes disperse in the cytoplasm. Since phosphorylation can alter protein-protein interactions and membrane traffic, we have examined the cell cycle-dependent phosphorylation of nuclear pore complex proteins. Nonmembrane nucleoporins Nup153, Nup214, and Nup358 that are modified by O-linked N-acetylglucosamine and recognized by a monoclonal antibody were phosphorylated throughout the cell cycle and hyperphosphorylated during M phase. Pore membrane glycoprotein gp210, that has a cytoplasmic, carboxyl-terminal domain facing the pore, was not phosphorylated in interphase but specifically phosphorylated in mitosis. Mutant and wild-type fusion proteins containing the cytoplasmic domain of gp210 were phosphorylated in vitro and their phosphopeptide maps compared to that of mitotic gp210. This analysis showed that Ser1880 of gp210 was phosphorylated in mitosis, possibly by cyclin B-p34cdc2 or a related kinase. Several nuclear pore complex proteins are therefore differentially phosphorylated during mitosis when pore complexes disassemble and reassemble.

tau Regulation of microtubule-microtubule spacing and bundling.

Tau proteins are microtubule-associated proteins that promote microtubule polymerization in vitro and in vivo. They are a family of neuronal proteins with apparent molecular weights in the range 50,000-68,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recently, a new member of this family has been described and its cDNA has been cloned. It has an apparent molecular weight of 116,000 and has been called high-molecular-weight tau (HMW tau). All the tau proteins are encoded by a single gene, which undergoes complex alternative splicing. In the present study, we have cloned into the baculovirus a cDNA fully encoding HMW tau as well as a truncated cDNA encoding a protein beginning 13 amino acids in front of the tau microtubule-binding domain. HMW tau-recombinant-virus-infected Sf9 cells overexpressed HMW tau, which induced the polymerization of microtubules and the formation of long cellular processes similar to those induced by low-molecular-weight tau (LMW tau) overexpression. Process cross sections revealed a larger spacing (approximately 35 nm) between microtubules when induced by HMW tau than when induced by LMW tau (approximately 20 nm). The truncated construct also induces processes, where microtubules were packed far more closely together (approximately 10 nm). Although branching did not occur in processes induced by intact tau S, 10% of the processes induced by the truncated tau protein branched.

Glycated tau protein in Alzheimer disease: a mechanism for induction of oxidant stress.

The stability of proteins that constitute the neurofibrillary tangles and senile plaques of Alzheimer disease suggests that they would be ideal substrates for nonenzymatic glycation, a process that occurs over long times, even at normal levels of glucose, ultimately resulting in the formation of advanced glycation end products (AGEs). AGE-modified proteins aggregate, and they generate reactive oxygen intermediates. Using monospecific antibody to AGEs, we have colocalized these AGEs with paired helical filament tau in neurofibrillary tangles in sporadic Alzheimer disease. Such neurons also exhibited evidence of oxidant stress: induction of malondialdehyde epitopes and heme oxygenase 1 antigen. AGE-recombinant tau generated reactive oxygen intermediates and, when introduced into the cytoplasm of SH-SY5Y neuroblastoma cells, induced oxidant stress. We propose that in Alzheimer disease, AGEs in paired helical filament tau can induce oxidant stress, thereby promoting neuronal dysfunction.

Cytoskeletons of central and peripheral neurons.

All eukaryotic cells have a cytoskeleton, consisting of microtubules, intermediate filaments and microfilaments. The cytoskeletal structure of cells and cell processes in the central nervous system is diverse. The generation of animal models in which specific mutations result in underexpression of overexpression of particular intermediate filament and microtubular proteins allows assessment of the possible role of cytoskeletal abnormalities in the neurodegenerative disorders. It is suggested that overexpression of filaments is likely to be the more significant process, but that neurofibrillary change, as recognized by the neuropathologist represents the final result of failure of any of a large number of molecular processes involved in cytoskeletal protein turnover.

Actin and neurofilament binding domain of brain spectrin beta subunit.

Tryptic digestion of brain spectrin generates a number of fragments from alpha and beta subunits; when these fragments are incubated with F-actin or neurofilament light subunit, four of them with molecular masses below 30 kDa sediment with the cytoskeleton structures. A selective purification of these fragments by ammonium sulfate fractionation and butyl-Sepharose chromatography has been achieved. Two fragments with molecular masses of 28 and 23 kDa bind to F-actin. Native brain spectrin causes half-maximal inhibition of the association at a concentration of 3 microM. Protein sequencing indicates that the actin-binding domain is contained in the beta subunit, in a stretch of amino acids at the N terminus from Ala43 (28-kDa fragment) or from Met104 (23-kDa fragment) and terminate probably at the C-terminal Lys288 or Lys284. Amino acids are numbered by reference to the sequence of the Drosophila beta subunit. The 28-kDa fragment also binds to the low-molecular-mass subunit of neurofilaments; brain spectrin heterodimer disrupts this binding. Hence, spectrin binds to F-actin and to neurofilaments via a common binding domain.

Interaction domains of neurofilament light chain and brain spectrin.

We have previously demonstrated that brain spectrin binds to the low-molecular-mass subunit of neurofilaments (NF-L) [Frappier, Regnouf & Pradel (1987) Eur. J. Biochem. 169, 651-657]. In the present study, we seek to locate their respective binding domains. In the first part we demonstrate that brain spectrin binds to a 20 kDa domain of NF-L. This domain is part of the rod domain of neurofilaments and plays a role in the polymerization process. However, the polymerization state does not seem to have any influence on the interaction. In the second part, we provide evidence that NF-L binds to the beta-subunit of not only brain spectrin but also human and avian erythrocyte spectrins. The microtubule-associated protein, MAP2, which has also been shown to bind to microfilaments and neurofilaments, binds to the same domain of NF-L as spectrin does. Finally, among the tryptic peptides of brain spectrin, we show that some peptides of low molecular mass (35, 25, 20 and 18 kDa) co-sediment with either NF-L or F-actin.

Immunological detection of spectrin during differentiation and in mature ciliated cells from quail oviduct.

A protein that was immunologically related to the erythrocyte and brain alpha-240-subunit and to the brain beta-235-subunit of spectrin was characterized by immunoblotting and was detected by immunofluorescence in the apical part of ciliated cells from quail oviduct. After immunogold-labeling electron immunocytochemistry, spectrin was detected mainly in a fibrillar meshwork located between the proximal parts of the basal bodies. It was also observed to be in contact with the basal foot of basal bodies, but was not found to be associated with the apical plasma membrane. Cilia and microvilli were unlabeled. In contrast, spectrin was detected in close contact with the lateral plasma membrane of mature ciliated cells as well as in stem epithelial cells in unstimulated oviduct. During ciliogenesis induced by estrogen, spectrin gradually appeared at the apex of the cells as the apical cytoskeleton differentiated.

Binding of brain spectrin to the 70-kDa neurofilament subunit protein.

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/50 +/- 10 mol NF-L and an apparent dissociation constant Kd = 4.3 x 10(-7) M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 x 10(-6) M with 1 mol fodrin bound/80 +/- 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 x 10(-7) M and the maximum binding was 1 mol fodrin/55 +/- 10 mol GFP.