Modulated induction of tau proteins in cultured human neuroblastoma cells.

Previous studies indicated that a chemically-defined, differentiation medium (DM) induces neuroblastoma cells, especially IMR32K cells, to exhibit phenotypes of mature neurons (including neurite outgrowth and synthesis of neurofilament polypeptides) and develop certain attributes of the neurons which are affected by neurofibrillary degeneration in Alzheimer's disease, such as expression of tangle-associated epitopes and accumulation of paired helical filaments-(PHF-) like fibrils. Immunocytochemical staining suggested that this cytoskeletal abnormality most likely results from altered expression of tau proteins. In the current study, we addressed this issue by analyzing tau-enriched preparations of IMR32K cells that were previously exposed to different incubation media using a panel of antibodies specific to tau and related microtubule-associated proteins. These cultured cells exhibited three groups of tau immunoreactivities which differ in molecular weight. Among them the level of high molecular weight tau (MW 90-112 kDa) was selectively augmented after DM incubation. The tau proteins produced in these neuron-like cells shared phosphorylated sites with PHF-tau and fetal tau, but differed from PHF-tau in their lack of the N-terminal insert which characterizes adult isoforms.

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.

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.

Characterization of a PC12 cell sub-clone (PC12-C41) with enhanced neurite outgrowth capacity: implications for a modulatory role of high molecular weight tau in neuritogenesis.

To address the means by which diversity of neuronal morphology is generated, we have isolated and characterized naturally occurring variants of rat PC12 pheochromocytoma cells that exhibit altered neurite outgrowth properties in response to nerve growth factor (NGF). We describe here a PC12 cell sub-clone, designated PC12-clone 41 (PC12-C41), that displays significant increases in neurite abundance and stability when compared with the parental line. This difference does not appear to be due to an altered sensitivity or responsiveness to NGF or to a more rapid rate of neurite extension. Because of the role of the cytoskeleton in neuritogenesis, we examined a panel of the major cytoskeletal proteins (MAP 1.2/1B, beta-tubulin, chartins, peripherin, and high and low molecular weight (HMW and LMW) taus) whose levels and/or extent of phosphorylation are regulated by NGF in PC12 cultures. Although most cytoskeletal proteins showed little difference between PC12 and PC12-C41 cells (+/- NGF treatment), there was a significant contrast between the two lines with respect to tau expression. In particular, while NGF increases the total specific levels of tau in both cell types to similar extents (by about twofold), the proportion comprising HMW tau is threefold higher in the PC12-C41 clone than in PC12 cells. A comparable difference was observed under substratum conditions that were non-permissive for neurite outgrowth and so this effect was not merely a consequence of the differential neuritogenic capacities of the two lines. The distinction between the expression of HMW and LMW taus in PC12 and PC12-C41 cells (+/- NGF) was also observed at the level of the messages encoding these proteins. Such findings indicate that initiation of neurite outgrowth in PC12 cultures does not require a massive induction of tau expression and raise the possibility that HMW and LMW taus may have differential capacities for modulating neuronal morphology.

Expression of high molecular weight tau in the central and peripheral nervous systems.

Using a novel PCR approach, we have cloned a cDNA encoding the entire high molecular weight tau molecule from rat dorsal root ganglia. The resulting 2080 bp cDNA differs from low molecular weight rat brain tau by the insertion of a novel 762 bp region (exon 4a) between exons 4 and 5. This cDNA clone is identical in sequence with a high molecular weight tau (HMW) cDNA from rat PC12 tumor cells and is closely related to a HMW tau cDNA from mouse N115 tumor cells. In vitro transcription/translation produces a protein that migrates on SDS-PAGE with the same apparent molecular weight as HMW tau purified from rat sciatic nerve. The HMW tau protein is generated from an 8 kb mRNA, which can be detected by northern blots in peripheral ganglia, but not in brain. A more sensitive assay using PCR and Southern blot analysis demonstrates the presence of exon 4a in spinal cord and in retina. In combination with immunohistochemical studies of spinal cord, these data suggest that HMW tau, though primarily in the peripheral nervous system, is also expressed in limited areas of the central nervous system, although its presence cannot be detected in the cerebral cortices.

Primary structure of high molecular weight tau present in the peripheral nervous system.

The tau proteins are a family of brain microtubule binding proteins that are required during axonal outgrowth and are found in neurofibrillary tangles in Alzheimer disease. A protein of higher molecular weight, immunologically related to tau, is expressed in the adult peripheral system and in cultured neuronal cell lines of neural crest origin. The predicted amino acid sequence of the high molecular weight tau from N115 cells has been determined from the sequence of its 2340-base-pair cDNA. High molecular weight tau contains an open reading frame encoding 733 amino acid residues. It contains sequences homologous to those present in the N-, middle, and C-terminal domains of adult brain tau proteins, including four homologous repeats, which are the tubulin binding sites, and an amino acid stretch, which is present only in the N-terminal domain of the mature brain variants. The middle region contains a previously unidentified nonhomologous stretch of 237 amino acid residues as well as a domain of 66 residues homologous to exon 6 of the bovine gene that is absent in all bovine, rat, and mouse tau cDNAs sequenced so far. A cDNA probe specific to the nonhomologous tau insert hybridizes to the 8- to 9-kilobase tau mRNA in N115 cells but not to the 6-kilobase tau mRNA in brain. Probes for the domains common to brain tau isoforms hybridize to both messages. The sequence of high molecular weight tau protein also suggests that it, like low molecular weight tau, is an elongated hydrophilic molecule. This cDNA should allow us to study the role of the domains specific to these tau forms in the specialization of the peripheral nervous system and for study of their expression in normal and pathological states.

High molecular weight tau: preferential localization in the peripheral nervous system.

Using epitope mapping we have demonstrated that a high molecular weight protein (Mr approximately 115 x 10(3)) present in brain and spinal cord is a member of the tau family of microtubule-associated proteins. Antibodies directed against the amino-terminal, middle and carboxyl-terminal portions of tau recognize this protein. A limited survey of neuronal tissues has shown that this high molecular weight tau protein is present in brain, spinal cord, dorsal root ganglia, dorsal and ventral roots and peripheral nerves. High molecular weight tau protein is expressed at higher levels in spinal cord than in brain and is the only form of tau detected in the adult peripheral nervous system.

Growth properties and biochemical characterization of mouse Schwann cells cultured in vitro.

Purified secondary cultures of mouse Schwann cells (less than 5% fibroblast contamination) have been obtained by taking advantage of the differential adhesion of Schwann cells and fibroblasts during trypsinization. The growth properties of the purified subcultures changed with time in culture. Cells passaged after 5 days in vitro (DIV) divided rapidly (doubling time 22 h), whereas cells that had been in vitro for longer periods progressively decreased their growth rate, becoming quiescent after 20 or more days. Schwann cells lacked the Thy 1.2 surface antigen, but were positively stained with antigalactocerebroside antibodies after prefixation. Biochemical analyses showed Schwann cells to be enriched in the activities of enzymes characteristic of the myelin-forming cells: 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP), cerebroside sulfotransferase (CST) and UDP-galactose: ceramide galactosyltransferase (CGalT).