Inhibition of Glutamate-induced Neurotoxicity by a Tau Antisense Oligonucleotide in Primary Culture...

European Journal of Neuroscience, Vol. 7, pp . 1603-1613, 1995 0 European Neuroscience Association

Inhibition of Glutamate-induced Neurotoxicity by a Tau Antisense Oligonucleotide in Primary Culture of Rat Cerebellar Granule Cells

Marina Pizzi, Alessandra Valerio, Virginia Arrighi, Paola Galli, Marco Belloni, Marina Ribola, Antonella Alberici, PierFranco Spano and Maurizio Memo Division of Pharmacology, Department of Biomedical Sciences and Biotechnologies, School of Medicine, University of Brescia, Italy

Keywords: neurodegeneration, cytoskeleton, phosphorylation, excitatory aminoacids

Abstract Short-term exposure of primary cultures of cerebellar granule cells from neonatal rat brain to high concentrations of glutamate resulted in a significant increase of both immunoreactivity to and mRNA levels of tau protein. Time- course experiments revealed the increases of tau immunoreactivity and mRNA levels to be maximal 2 h after the glutamate pulse. To investigate the relationship between newly synthesized tau protein and glutamate- induced neurotoxicity, neurons were preincubated with a specific tau antisense oligonucleotide. This treatment resulted in (i) inhibition of the glutamate-induced increase of tau immunoreactivity and (ii) a decrease in the sensitivity of the neurons to neurotoxic concentrations of glutamate. These data indicate that induction of the cytoskeleton-associated tau protein participates in the cascade of events promoted by glutamate leading to neurodegeneration.

Introduction Excitatory amino acid neurotransmission in the mammalian central nervous system is mediated principally by glutamate and structurally related compounds. It is estimated that glutamate is the primary neurotransmitter in -50% of the synapses in the mammalian forebrain. In fact, nearly all neurons can be excited by glutamate. In addition to its involvement in information coding, glutamate may also act as a neurotrophic factor by participating in the cytoarchitectural organization of neurons during brain development (McDonald and Johnston, 1990). These effects can be also detected in vitro. When tested on primary cultures of different types of neuronal cells during differentiation, glutamate through the stimulation of specific glutamate receptor subtypes induces the major stages of development, such as neurite extension and branching, and synaptogenesis (Balazs et al., 1989; Brewer and Cotman, 1989; Rashid and Cambray-Deakin, 1992; Pizzi et al., 1994a). In particular, the ability of glutamate to influence the structure of developing neurons and their pattern of inter- connectivity is mainly, although not entirely, related to its capacity to increase cytosolic calcium levels through stimulation of a specific subtype of ionotropic glutamate receptors, namely the N-methyl-D- aspartate (NMDA)-selective glutamate receptor.

Under certain undefined, adverse conditions, glutamate may become a potent excitotoxin (Rothman and Olney, 1988; Choi, 1988). Experi- ments performed both in vivo and in vitro indicated that excessive stimulation of glutamate receptors promotes a cascade of events leading to neuron death. This effect can be achieved by directly stimulating specific glutamate receptor subtypes, mainly the NMDA-

selective glutamate receptors, or indirectly by a number of either experimental manoeuvres or pathological conditions including transient ischaemia, glucose deprivation, seizures, or physical damage, which ultimately result in hyperactivity of glutamatergic neurons (Simon, 1992). In this regard, glutamate has been considered one of the mediators of the pathophysiology of several neurodegenerative disorders including stroke, Parkinson’s disease, Huntington’s chorea and Alzheimer’s disease (Choi, 1988; Benveniste et nl., 1988; Pomara et al., 1992; Lipton and Rosenberg, 1994). Several mechanisms have been proposed to explain glutamate’s transition from neurotrophic factor to neurotoxin, and it is still unclear whether the different responses of neurons to glutamate are mediated by a common mechanism.

Recent data by Mattson (1990) showed that exposure of primary cultures of rat hippocampal neurons to glutamate induced antigenic changes similar to those seen in neurofibrillary tangles (NFTs). The NFTs are one of the defining features of Alzheimer’s disease, although they may occur in other disease entities including subacute sclerosing panencephalitis, dementia pugilistica and Guamanian Parkinson dementia complex, as well as in normal ageing (Wisniewski et al., 1979; Amagada et al., 1992). These lesions are made of bundles of abnormal filaments, called paired helical filaments (PHFs), which accumulate in selective subpopulations of neurons within specific, highly vulnerable target areas (Kidd, 1963; Kosik, 1990). Accumula- tion of these PHFs is associated with disorganization of the normal cytoskeleton in affected neurons and fragmentation of the microtubule

Correspondence to: Prof. Maurizio Memo, Department of Biomedical Sciences and Biotechnologies, University of Brescia, Via Valsabbina, 19 25 124 Brescia, Italy

Received 3 August 1994, revised 26 September 1994. accepted 27 February I995

1604 Tau antisense decreases glutamate neurotoxicity

network. One of the major components of PHF is the cytoskeleton- associated tau protein (Brion et al., 1985; Goedert er al., 1988; Wischik et al., 1988), a neuronal protein promoting microtubule assembly and neurite polarization (Lee, 1990; Goedert et al., 1991a). In fact, tau proteins are a class of low molecular mass proteins which are shown to be closely related by amino acid composition (Lee et al., 1988; Kanai et al., 1989). Tau proteins show a development evolution and their expression seems relevant, although apparently not necessary, for the differentiation of neurites into axons (Kosik et al., 1989; Caceres and Kosik, 1990; Harada et al., 1994). Recent data also suggest tau polypeptides as more than neuronal microtubule- associated proteins in that they localize in the nuclei of dividing primate cells in culture (Loomis et al., 1990; Wang et al., 1993).

Since tau proteins are thought to be encoded by a single gene, it is considered likely that tau heterogeneity arises via both differential mRNA processing and post-translational modifications. Interestingly, PHF-tau are modified forms of tau proteins. In comparison with normal tau, PHF-tau proteins have higher molecular masses, lower isoelectric points and fewer isoforms, and react differentially to some anti-tau antibodies (Kosik et al., 1986; Greenberg and Davies, 1990; Liu et al., 1991). Several possible modifications have been proposed for PHF-tau that make them different from normal tau proteins. Of these, aberrant phosphorylation is generally accepted as one of the major changes in PHF-tau (Grundke-Iqbal et al., 1986; Ihara et al., 1986; Flament et al., 1990). The molecular mechanism(s) responsible for tau accumulation andor hyperphosphorylation. as well as the temporal hierarchy coupling NFT formation and neurodegeneration, has not been completely clarified. We now report that expression of the cytoskeleton-associated tau protein in primary cultures of cerebellar granule cells from neonatal rats is induced by glutamate in a dose- and time-dependent fashion. Moreover, the glutamate- induced increase of tau expression appears to be one of the key factors in dictating the glutamate-mediated intracellular programme leading to neuron death.

Materials and methods

Cell culture Primary cultures of cerebellar granule cells were prepared from 8-day-old Sprague-Dawley rat pups as previously described (Pizzi et al., 1991). Cells were plated onto poly-L-lysine-coated dishes and cultured in basal Eagle’s medium containing 10% heat- inactivated fetal bovine serum, 2 mM glutamine, 50 pg/ml gentamicin and 25 mM KCI, at the density of 1.5 X lo5 cells/cm2. Cytosine arabinoside (10 pM) was added to the cultures 18 h after seeding to prevent non-neuronal cell proliferation. Experiments were done after culturing the neurons for 10-12 days.

Neurotoxicity assay The culture-conditioned medium was collected and the cells were washed once with Locke’s solution (154 mM NaCI, 5.6 mM KCl, 3.6 mM NaHC03, 2.3 mM CaCI2, 5.6 mM glucose, 5 mM HEPES, pH 7.4) supplemented with 10 pM glycine, and exposed to different concentrations of glutamate for 15 min. After this period, cells were

washed three times with Locke’s solution containing 1 mM MgS04 and returned to the culture-conditioned medium. Tau sense or antisense oligonucleotide was added to the cell culture medium 1 h before the glutamate pulse. Cells were cultured for an additional 24 h before intravital staining was performed. Cell viability was established according to Jones and Senft (1985). Briefly, cells were washed with Locke’s solution and stained for 3 min at 22°C with a mixture of

fluorescein diacetate (15 pg/ml) and propidium iodide (80 pg/ml). The stained cells were examined immediately with a standard epi- illumination fluorescence microscope (450 nm excitation, 520 nm barrier). Fluorescein diacetate crosses the cell membranes and is hydrolysed by intracellular esterase to produce green-yellow staining. This process is reduced by neuronal injury, a condition facilitating propidium iodide penetration and interaction with DNA to yield a red fluorescence. The percentage of surviving neurons in the monolayer was computed by calculating the ratio between the fluorescein diacetate and fluorescein diacetate plus propidium iodide stainings in photomicrographs of at least three representative fields from each monolayer.

lrnrnunostaining Tau immunoreactivity was measured 2 h or 4 h after the glutamate pulse according to a previously described procedure (Mattson, 1990; Pizzi et al., 1993). Briefly, cultures were fixed for 30 rnin in 50 mM phosphate-buffered saline (PBS) containing 4% paraformaldehyde. Following several rinses in PBS, cells were incubated for 20 rnin in Tris-buffered saline containing 0.5% hydrogen peroxide to block endogenous peroxidase staining. Cells were then permeabilized by a 5 min exposure to 0.2% Triton X-100 in PBS and incubated for 24 h at 8°C with the primary antibody. The primary antibodies used in the present study were (i) anti-tau monoclonal antibody Alz-50 ( I : 100 dilution), kindly supplied by P. Davies, (ii) anti-tau monoclonal antibody TAU-2 ( 1 : 1 ,OOO dilution) from Sigma, and (iii) anti-MAP2 monoclonal antibody (l:5OO dilution), from Sigma. The specificity of the antibodies has been described previously (Wolozin et al., 1986; Papasozomenos et al., 1987; Turker, 1988). After rinses, cells were processed using the avidin-biotin complex kit (ABC Elite kit, Vector Laboratories). Enzymatic reaction was developed as described by Mattson (1990). In order to verify the specificity of the chromogen reaction, some dishes were processed identically except that cells were incubated with the primary antibody solvent. In these conditions, no immunostaining was generated.

A blind analysis of immunoreactive cells was performed in all dishes. A minimum of 100 neurons were counted in at least three fields of three different culture dishes. A semiquantitative study of immunoreactivity was camed out using the Magiscan Image Analysis System, designed and made by Joyce-Loebl Ltd (London, UK). The General Image Analysis Software (GENIAS) provided by Joyce- Loebl as standard with Magiscan gave us access to the image processing and analysis function of Magiscan. The microdensitometry analysis was performed as previously described (Mize et al., 1988) by measuring the following parameters in each cell body: (i) integrated opical density (ID), i.e. the sum of optical density for each pixel in the cell area considered; and (ii) area, i.e. the sum of pixels over the image of the cell pointset. The ratio ID/area from each cell was processed for the statistical analysis. The statistical significance of differences between the values was tested by analysis of variance. Data are presented as the mean 2 SEM of at least three experiments.

Western blot analysis Ten million cells were harvested in 100 pI of 0.1 M PIPES buffer, pH 6.9, containing 1 mM phenyl methyl sulphonyl fluoride, 1 pglml leupeptin, 5 pg/ml aprotinin and 1 pglml pepstatin. The suspension was sonicated for 30 s at full power and centrifuged at 15 OOO g for 30 min at 4°C. The resulting supernatant was isolated and protein content determined by a conventional method (BCA protein assay kit, Pierce, Rockford, IL). Fractionation of microtubule-associated proteins was performed according to Herzog and Weber (1978) with minor modifications. Briefly, NaCl and dithiothreitol were added to

Tau antisense decreases glutamate neurotoxicity 1605

in the presence of [ 32P]dCTP as recommended by the manufacturer. The resulting cDNA was quantitated by determining the amount of radioactivity incorporated into trichloroacetic acid-precipitable nucleic acids. PCR was done with Taq DNA polymerase (Perkin Elmer Cetus) in 100 pl of standard buffer containing 0.5 ng cDNA, [32P]dCTP as radioactive tracer and 1 pM specific primers. The following primers were used: 5'-AGCCGCCTACAGACTGCC- CCTGTG-3' corresponding to nucleotides 693-7 17 and referred to as A, and 3'-ATTTCTGCTCCATGGTCTGTCITG-5', corresponding to nucleotides 1124-1 147 and referred to as B, of the tau cDNA sequence (Kosik et af., 1989). Thirty cycles of amplification were performed with a DNA Thermal Cycler (Perkin Elmer Cetus) and a step programme (94"C, 30 s; 60"C, 30 s; 72°C. 60 s), followed by a 15 min final extension. PCR products were separated by electrophoresis, visualized by ethidium bromide staining and cut out. The radioactivity incorporated into the bands was counted by scintillation spectrometry.

For quantitative adaptation of the PCR method an internal standard was introduced with the sample into the PCR reaction mixture. The internal standard was synthesized according to Celi et al. (1993). Briefly, the PCR product corresponding to the short tau isoform was cut out of the gel, purified, and used as a template for PCR using primer A and a second primer 48 nucleotides in length in which 24 nucleotides at the 3' end corresponded to nucleotides 1002-1026 of the target sequence and 24 nucleotides at the 5' end corresponded to primer B. The PCR product was separated by electrophoresis, visualized by ethidium bromide, cut out, and quantitated by conventional methods. A known number of copies of the internal standard (corresponding to pg) was introduced into the samples and amplified with primers A and B. With this approach, three products were generated, two derived from the endogenous template corresponding to the long and short tau mRNA isoform, and one corresponding to the internal standard.

Incorporation of tau antisense oligonucleotide into granule cells Either tau sense or antisense oligonucleotide was end-labelled with y-[32P]ATP (New England Nuclear) and bacteriophage T4 polynucleo- tide kinase and purified by polyacrylamide gel electrophoresis, and the eluted sample was ethanol-precipitated and resuspended in medium. An activity of 2X106 c.p.m. was added to the culture medium with unlabelled oligonucleotide up to 5 pM concentration. At various times after the addition of the oligonucleotide, the culture medium was removed and saved, and cells were washed once. The washing buffer was rapidly removed and saved, and cells were lysed in Tris-HCI buffer, pH 7.4, containing 1% sodium dodecyl sulphate, and DNA/RNA was extracted with 0.1 ml of phenol. Radioactivity was counted both in the culture medium and in DNA/RNA extracts. To determine the stability of the oligonucleotide, aliquots of nucleic acid extracts and culture media were electrophoresed on denaturating 20% polyacrilamide gels, and gels were exposed to Kodak AR film for 24 h. Values (mean 2 SEM of five or six determinations) represent the radioactivity taken up by the cells as a percentage of the total radioactivity added to the corresponding culture dishes at different periods of time after the addition of the oligonucleotide.

the protein extracts to a final concentration of 1 M and 10 mM respectively. Protein solutions were rapidly pipetted into preheated Sorvall Corex glass tubes and kept in a boiling water bath for 5 min. Tubes were then rapidly placed on ice and centrifuged at 15 000 g for 30 min at 4°C. Supernatants were collected and dialysed 16 h using a Spectra/Por membrane (6000-8000 molecular weight cutoff). Aliquots of heat-resistant, microtubule-associated proteins corresponding to 50 pg of total protein in the original extracts were electrophoresed on 10% sodium dodecylsulphate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose paper (Schleicher and Schuell, Dassel, Germany). Filters were incubated at 4°C over- night with a polyclonal anti-tau antibody (Sigma) diluted 1:100 in 3% fat dried milk (Sigma). For immunodetection, a goat anti-rabbit alkaline phosphatase conjugate antibody (Promega), 1 :7500 dilution, was used. Densitometric analysis was done using LKB 2222-020 UltroScan XL Laser Densitometer.

Measurement of intracellular calcium concentration Regulation of cytosolic free calcium concentration ([Ca2+]i) by glutamate in control and tau antisense-treated cells was investigated by microfluorimetry in single cerebellar granule neurons according to Malgaroli et al. (1987) with minor modifications. Cells were plated on 100 pg/ml poly-L-lysine-coated glass coverslips and cultured as described above. At 1&14 days in vitro, cells were loaded with the calcium-sensitive fluorescent dye fura-2, by a 60 min incubation at 37°C with its acetoxymethyl derivative (4 pM in Krebs-Ringer medium containing 1.3 mg/ml bovine serum albumin, pH 7.4) and then mounted in a 22 mm holder that created a chamber with the coverslip on the bottom. Fura-2 emission was monitored using an inverted fluorescence microscope (Nikon Diaphot) associated with an intensified charge-coupled device camera (MIRA- 100 TE; Applied Imaging, Gateshead, UK) which recorded the 510 nm fluorescence emission in neurons excited through a narrow bandpass filter (340 nm; 380 nm). The background was subtracted and the amount of free calcium within the cell bodies was calculated from the ratio of 340/380 nm obtained every 3-4 s. Calibration was according to external standards of calcium and fura-2 (Connor, 1986). Fluorescence image acquisition and analysis were performed using the MIRAcal (Multiple Image Ratioing and Analysis with Calibration) system by Applied Imaging.

Tau antisense oligonucleotide synthesis Both sense and antisense tau oligonucleotides were synthesized on an Applied Biosystems 391 DNA synthesizer, using phosphor- amidite chemistry. Oligonucleotides were purified by reverse-phase chromatography using Oligo-Pak oligonucleotide purification columns (Milligen Biosearch, Foster City, CA) following the recommenda- tions of the manufacturer. The tau antisense oligonucleotide was 26 nucleotides long and comprised the ATG codon corresponding to the initiator methionine. The tau antisense oligonucleotide sequence was 5'-GTTCAGCCATGCTGCTTCAAAGCCTG-3', corresponding to nucleotide - 16 to nucleotide + 10 of the rat tau gene sequence (Kosik et al., 1989). The sense oligonucleotide was the exact inverse complement of the antisense oligonucleotide.

Polymerase chain reaction A polymerase chain reaction (PCR)-derived method was used to quantitate tau mRNA levels. At the end of the incubation, cells were harvested in guanidium thiocyanate. Total mRNA was isolated by centrifugation, extracted by chloroform+thanol and reverse-transcribed with Moloney murine leukaemia virus reverse transcriptase

Results Glutamate-induced increase on tau immunoreactivity Exposure of primary cultures of neonatal rat cerebellar granule cells to concentrations of glutamate ranging from 10 to 500 pM for 15 min in a Mg2+-free solution results in concentration-dependent


cell death which is easily detectable 24 h after the pulse (Fig. 8). In our experimental conditions, the maximal neurotoxic effect was reached when glutamate was added at the concentration of 100 pM. We previously found that a brief pulse of glutamate induced accumulation of tau immunoreactivity in primary cultures of cerebellar granule cells (Pizzi et al., 1993). In the present study we extended the immunological characterization of this effect. Particularly, immunocytochemistry for tau proteins was carried out with the anti- tau monoclonal antibody TAU-2, which recognizes both phosphorylated and non-phosphorylated tau isoforrns, and with Alz-50, a mouse monoclonal antibody raised against Alzheimer’s disease brain homogenates. We found that a 15 min application of I 0 0 pM glutamate to cerebellar granule cells induced a significant increase in the level of tau immunoreactivity, which reached a maximum 2 h after the glutamate pulse. The results from a representative immunostaining are in Figure IA-D. A mild TAU-2 and Alz-50 immunostaining (Fig. IA and C respectively) was observed in cell bodies and neurites of untreated cells. Exposure of the cultures to 100 pM glutamate for 15 min increased TAU-2 and Alz-50 immunoreactivity in cell bodies of the neurons (Fig. 1B and D respectively). Semi- quantitative analysis of neuronal somata immunolabelling was carried out by a computer-assisted system as described in Materials and

methods. As shown in Figure 2, glutamate treatment increased tau immunoreactivity in most of the neurons, as revealed by the shift of cell grey distribution towards higher levels.

The glutamate-induced increases of TAU-2 as well as of Alz-50 immunoreactivity tended to return to basal levels in the next 2 h and were evoked, although with less efficacy, by subtoxic (10 pM) concentrations of glutamate (data not shown). A dose-dependent increase of TAU-2 immunostaining induced by glutamate has previously been found in primary culture of rat cortical cells (Sindou et al., 1992).

The increases in TAU-2 and Alz-50 immunostaining induced by glutamate appeared as the result of specific changes in antigenicity. In fact, immunostaining with anti MAP-2 antibody was not noticeably altered by the glutamate treatment. The results from a representative

FIG. 2. Semiquantitative analysis of tau and MAP2 immunoreactivity in rat cerebellar granule cells untreated (left side) or exposed for 15 min to 100 pM glutamate (right side). Cells were immunostained with anti-tau monoclonal antibody TAU-2 (upper panel), anti-tau monoclonal antibody Alz-50 (middle panel), or anti-MAP2 monoclonal antibody (lower panel). Columns represent the mean 2 SEM of the percentage of cells for each level of grey, expressed

FIG. I . lmmunocytochemical analysis of tau and MAP2 protein in cerebellar as integrated density/area. At least 100 cells taken from three dishes were granule cells. Micrographs are bright-field views with monoclonal anti-tau examined in three separate experiments. Integrated density is the sum of antibody TAU-2 (A, B), monoclonal anti-tau antibody Ah-50 (C, D), and optical density (OD) for each pixel in the cell area considered; the area monoclonal anti-MAP-2 antibody (E, F) of untreated (A, C. E) or glutamate- represents the sum of pixels in the image over the cell body. Grey levels treated (B, D, F) cells. Staining was done 2 h after the glutamate (100 FM) are expressed by a defined score: A = 0-5 x OD/pixel; B = pulse with the ABC Elite Vector kit after paraformaldehyde fixation. Bar = 5-7 X 15 pM. OD/pixel; E = 11-15 X

OD/pixel; C = 7-10 X OD/pixel; D = 7-11 X OD/pixel; F = 15-20 X lo-’ ODlpixel.


experiment and the computerized immunostaining quantitation are reported in Figures IE-F and 2 respectively.

Western blot analysis of tau proteins Change in tau protein levels after glutamate application were also investigated by Western blot analysis. A representative immunoblot of heat-stable protein extracts from untreated cultures and cultures exposed to glutamate immunostained with anti-tau polyclonal antibody is shown in Figure 3. Anti-tau antiserum clearly recognized a set of five bands migrating in the range from 50 to 62 kDa in protein extracts from untreated cultures (lane I). In particular, the anti-tau antibody detected a predominant band of 62 kDa and four sharp bands of 61, 59, 56 and 54 kDa respectively. Analysis of protein extracts from granule cells 2 h after exposure to 100 pM glutamate for 15 min resulted in an increase of tau immunoreactivity mainly affecting two bands with apparent molecular weights of 59 and 54 kDa, without changes in the electrophoretic migration pattern of the bands (lane 2). The glutamate-induced increase of tau protein level reached a maximum 2 h after the pulse, was still present at 4 h and was virtually undetectable 8 h after the pulse. Densitometric analysis of the immunoblot shown in Figure 3 revealed a net increase of two- to three-fold in the 59 and 54 kDa immunostained bands in lane 2, in comparison with those of lane 1 (data not shown).

FIG. 3. Representative Western blot analysis of protein extracts from cerebellar granule cells immunostained with anti-tau polyclonal antibody. Lane 1 , untreated cultures; lane 2, cultures treated with 100 pM glutamate; lane 3, cultures treated with 25 pM tau antisense oligonucleotide; lane 4, cultures treated with both 100 pM glutamate and 25 pM tau antisense. Cells were harvested 2 h after the 15 min glutamate pulse. Thermostable fractions corresponding to 50 pg total cellular proteins were loaded on each well of a 10% sodium dodecyl sulphate-polyacrylamide gel. Samples from glutamate- treated cultures are indicated with +. Size of molecular weight markers is indicated on the left. Arrow heads note two bands with apparent molecular weight of 59 and 54 kDa.

Glutamate-induced increase of tau mRNA levels A quantitative PCR-derived method was applied to evaluate tau mRNA content in primary cultures of cerebellar granule cells. The two primers used for PCR were specifically designed to amplify a region of the rat tau mRNA encoding the COOH-terminal domain of tau protein (Fig. 4A). The selected sequence was 455 nucleotides long and contained four internal repeats of 93 nucleotides each. PCR done in the presence of the tau-specific primers and reverse-transcribed mRNA from granule cells yielded two distinct DNA bands corresponding to -360 and -455 bp. The long PCR product corresponds to that theoretically expected on the basis of tau sequence, while the short one may reflect tau mRNA species lacking one internal 93 bp repeat. This was proved by sequencing both PCR products.

To allow the relative quantitation of different mRNA levels by PCR, a series of preliminary experiments was done to correlate the effects of different concentrations of cDNA from granule cells with the yield of PCR products. The amount of radioactivity incorporated into the DNA-amplified bands was proportional to the amount of reverse-transcribed mRNA in a range between 0.1 and 2.0 ng. Thus, for the most accurate quantitation, 0.5 ng of cDNA was used for comparing the tau cDNA-amplified products from different cell culture samples. Finally, a DNA segment identical to the short tau mRNA species lacking 98 internal base pairs was used as internal standard. A schematic representation of internal standard synthesis and the results from a representative experiment are shown in Figure 4B and C respectively. Details are in Materials and methods. A known number of copies of the internal standard was routinely introduced to each sample and served to evaluate possible inter- and intra-assay variations in PCR efficiency between different experimental samples.

Using this approach, we found that application of glutamate to primary cultures of cerebellar granule cells for 15 min induced a time- and concentration-dependent accumulation of both tau mRNA species (Fig. 5 ) . Particularly when measured 2 h after the pulse, glutamate induced half-maximal and maximal effects at concentrations of 7 and 60 pM respectively (Fig. 5A). It is noteworthy that glutamate showed higher affinity in inducing tau gene expression than in eliciting neurotoxicity. Indeed, tau mRNA levels were significantly increased even by a concentration of glutamate that is inactive in producing cell death, i.e. 10 pM. Tau mRNA levels were also measured at different times after application of either toxic (100 pM) or subtoxic (10 pM) concentrations of glutamate (Fig. 5B). The time- course experiments showed that the maximal accumulation of tau mRNA occurred 2 h after the glutamate pulse. At this time, the increase of tau mRNA level induced by 100 pM glutamate was almost twice that induced by 10 pM glutamate. The data reported in Figure 5B, which refer to the long tau mRNA isoform, show that glutamate induced increases of 200 and 350% in tau mRNA accumulation at subtoxic and toxic concentrations respectively. Four hours after the glutamate pulse tau mRNA levels decreased, tending to basal values. Data obtained measuring the relative abundance of the short tau mRNA isoform were superimposable onto those shown in Figure 5B (data not shown).

Glutamate-induced tau immunoreactivity in tau antisense oligonucleotide-treated cells. An oligonucleotide complementary to a 26 nucleotide sequence comprising the ATG translation initiation codon of the tau gene, and its exact inverse complement, the sense oligonucleotide, were synthesized and added at the concentration of 25 pM to the cultured neurons 1 h before the glutamate pulse. The antisense oligonucleotide


3 -

-

2 -

-

1 -

-

a) 5' --+ LONG ISOFORM (Tau 4r)

$ . $ . SHORT ISOFORM (Tau 3r)

n cDNA - 7 - 7

B PCR A

+ --- Tau4r

Tau 3r

c+1B\ I

A

PCR - INTERNAL STANDARD (Inl. SI.)

7 __ A- B

FIG. 4. Schematic representation of rat tau cDNA sequence (a), procedure for internal standard synthesis (b), and results from a representative PCR experiment (c). (a) Solid boxes are the internal repeats, white boxes are sites of differential splicing. Arrows indicate the position of the oligonucleotide primers (A and B) used for PCR. (b) A, B and C+B indicate the oligonucleotides used for the synthesis of the tau internal standard. Details of sequence and technical procedures are in Materials and methods. (c) Arrows indicate positions of 455 and 360 bp bands, which correspond to the long, mature and the short, juvenile tau isoform respectively, and the position of the 262 bp band, which corresponds to the internal standard. Lane I , DNA ladder; lane 2, cDNA from granule cells (0.5 ng); lane 3, internal standard pg); lane 4, coamplification of cDNA from granule cells and internal standard (same amounts as before); line 5, no cDNA and no internal standard.

i d / GI A

0-I-P- 0 10 50 100 500

Glutamate concentration (pM)

B

1

0-I-P- 0 1 2 3 4 5

Time after glutamate pulse (h)

FIG. 5 . Relative abundance of tau mRNA levels. (A) Dose-response relationship. Points are from the data obtained measuring the relative abundance of the short, juvenile (filled circles) and the long, mature (open circles) tau isoform 2 h after a glutamate pulse. (B) Time-course. Cells were exposed to 10 pM (filled circles) or I 0 0 pM (open circles) glutamate for 15 min. Points are from the data obtained measuring the relative abundance of the long. mature tau mRNA isoform. Values in panels A and B are expressed as the ratio between the radioactivity incorporated into the PCR products derived from endogenous tau mRNA and the radioactivity incorporated into the PCR product from the exogenous internal standard. Values represent the mean 2 SEM of five or six determinations.

is believed to form RNA-DNA hybrids with the endogenous sense sequence, thus reducing efficiency of translation, stability or transport of the mRNA concerned (Eguchi ef al., 1991). The experimental protocol used in the present study, including oligonucleotide sequence and concentration, was similar to that established by Caceres and Kosik (1 990) for demonstrating the inhibition of neurite polarity by tau antisense oligonucleotides in primary cultures of cerebellar neurons.

First, an attempt was made to demonstrate that the oligonucleotide

entered the cells. As shown in Figure 6, the 32P-labelled tau antisense oligonucleotide was found within the neurons already 15 min after application. The amount of radioactivity incorporated by the cells increased with time, reaching a plateau at 1 h. An acrylamide gel of the DNARNA cell extracts at the same time intervals revealed that nearly all the labelled oligonucleotide inside the cells remained intact after passage through plasma membranes (data not shown). The pattern of incorporation of the labelled sense oligonucleotide by the


0 15 30 60 time (min.)

120

FIG. 6. Uptake of "P-labelled tau antisense (filled circles) and sense (open circles) oligonucleotide by cerebellar granule cells. Values (mean 2 SEM of five or six determinations) represent the fraction of radioactivity taken up by the cells over the total radioactivity added to the corresponding culture dishes at different periods of time after the addition of the oligonucleotide, and are expressed as percentages.

Control

- Q 0 c c

C

Tau-As

A B C Glutamate

A B C A B C Glutamate

FIG. 7. Semiquantitative analysis of tau antisense (Tau-As) and vehicle (control) treatments on TAU-2 immunoreactivity in cerebellar granule cells exposed or not to I 0 0 pM glutamate. Cells were pretreated with vehicle or 25 pM tau antisense oligonucleotide I h before the 15 min glutamate pulse. Staining was done 2 h after the glutamate pulse. Semiquantitative analysis was performed as described in legend of Figure 2. Columns represent the mean 2 SEM of the percentage of cells for each level of grey in three dishes of two separate experiments.

cells was similar to that of the antisense and both were not affected by the glutamate pulse.

We then evaluated the efficacy of the tau antisense oligonucleotide

treatment. As reported in Figure 7, pretreatment of granule cells with tau antisense oligonucleotide completely prevented the increase of TAU-2 immunoreactivity induced by glutamate, while it did not significantly affect basal TAU-2 immunostaining. The effect was specific since pretreatment of the cells with the same concentration of the sense oligonucleotide did not alter the capability of glutamate to increase TAU-2 immunoreactivity (data not shown). Western blot analysis of microtubule-enriched protein extracts from untreated cultures, cultures exposed to tau antisense alone, cultures exposed to glutamate alone, and cultures exposed to tau antisense plus glutamate, further shows that tau antisense oligonucleotide treatment results in the inhibition of the glutamate-induced tau protein levels, without affecting basal tau immunostaining (Fig. 3, lanes 3 and 4).

In a previous series of experiments, we found that addition of different concentrations (from 10 to 50 pM) of tau oligonucleotide antisense for 24 h to cultures of cerebellar granule cells at 3 days in vitro induced a sustained reduction both of TAU-2 immunoreactivity and of neurite growth. The maximal effect was obtained with 50 pM tau oligonucleotide antisense, and resulted in the complete disappearance of TAU-2 immunoreactivity associated with no change in MAP-2 immunoreactivity. The same time of exposure and range of concentrations did not alter either TAU-2 immunoreactivity or neurite extension in cultures of cerebellar granule cells at 12 days in vitro, a time at which they are morphologically mature with established branches and neurites (Pizzi et al., 1994b). The concentration of tau oligonucleotide antisense used in the present study, 25 pM, was thus specifically chosen because of its efficacy in conditions in which the expression of tau proteins is extremely elevated. The greater sensitivity of tau antisense oligonucleotides of differentiating cells has been previously shown by Caceres and Kosik (1990) in cerebellar neurons and by Hanemaaijer and Ginzburg (1991) in PC12 cells treated with nerve growth factor.

Glutamate-induced tau neurotoxicity in tau antisense oligonucleotide-treated cells Pretreatment of granule cells at 12 days in vitro with either sense or antisense oligonucleotides for 1 h did not modify the viability or the gross morphology of the cells for at least 48 h (Pizzi et al., 1993); however, the response to neurotoxic concentrations of glutamate was significantly changed. As depicted in Figure 8, the concentration- response curve of glutamate for inducing neuronal death in cells pretreated with tau antisense oligonucleotide showed a shift to the right when compared to those obtained in untreated or in sense oligonucleotide-treated cells. In particular, the EC50 values of glutamate for inducing cell death were 20 and 100 pM for sense-treated and antisense-treated cells respectively. The EC50 value of glutamate for inducing neurotoxicity in vehicle-treated cells was 25 5 5 pM. The neurotoxic effects induced by the maximally effective concentration of glutamate, i.e. 500 pM, were not affected by either sense or antisense treatment.

Glutamate-induced rise of [&?+Ii in tau antisense oligonucleotide-treated cells Rat cerebellar granule cells used for [Ca2+]i recording were cultured in vitm for a period of 10-14 days. A small recording field (70 mm2) was specifically employed in our experiments to restrict the evaluation to the changes occurring in the cell body. As shown in Figure 9, panel C, [Ca2+Ii of resting granule cells was 40 5 10 nM (n = 25), a value similar to those previously reported (Ciardo and Meldolesi, 1991; Fohrman et al., 1993). Glutamate at 50 pM evoked a rapid [Ca2+Ii rise (Fig. 9A) followed by a sustained


100

.- E

B e 5 0 a

0 X .-

al c

100

.- E

B e 5 0 a

0 X .-

al c 0 c

8

0

c 0 8

0 10 50 100 500

Glutamate concentration (pM)

FIG. 8. Dose-dependent neurotoxic effects of glutamate in primary cultures of cerebellar granule cells pretreated 1 h before with vehicle (open circles), tau sense oligonucleotide (filled circles) or tau antisense oligonucleotide (open squares). Values represent the percentage of dead cells 24 h after the glutamate pulse. Data (mean 2 SEM of five or six determinations) are representative of a typical experiment repeated with similar results in at least three preparations of neurons. Viability of vehicle-treated control cultures was 88 2 7% and did not significantly differ either from tau sense or antisense oligonucleotide- pretreated cultures. * P < 0.0 I versus the coresponding glutamate concentration value in vehicle-treated cells.

plateau (650 2 10 nM) in all the neurons investigated ( n = 30). heincubation of the cells with 25 pM tau antisense oligonucleotide for 1 h did not alter the [Cazfli responsiveness of the neurons to glutamate. As shown in Figure 9B, in the tau antisense-treated cells ( n = 31) glutamate induced a rapid [Ca2+Ii rise followed by a plateau, with kinetics and intensity similar to those detected in untreated cells.

Discussion Primary cultures of cerebellar granule cells from neonatal rat brains express different species of mRNAs derived by alternative splicing of the mRNA encoding tau proteins. This is based on the results obtained by evaluating tau mRNA content with a PCR-derived method. Indeed, by using two primers specifically designed to flank the repeat-containing domain of the tau gene, it was possible to determine the presence of two distinct amplified DNA PCR products. Heterogeneity of tau protein derived from differential mRNA splicing has been described already (Lee et al., 1988; Kosik et al., 1989). In particular, it has been reported that there are two tau isoforms which are distinguishable by the presence of four and three internal repeats respectively. In the rat, the low molecular weight form is expressed in early postnatal brain while the high molecular weight form is expressed in mature brain (Kosik ef al., 1989). On the basis of their different patterns of expression during development, they have been named as ‘mature’ and ‘juvenile’ tau mRNA isoforms. We adopted this nomenclature, describing as mature and juvenile tau mRNA isofoms those reflected by the 455 and 360 bp PCR products respectively.

In the rat, the shift to mature tau expression is approximately at postnatal day 8, which is coincident with the age of the rat used in our study. Since at that time cerebellar granule cells are still undifferentiated, they may be supposed to express preferentially, if not exclusively, the juvenile isoform of tau. However, in our experimental

1000,

Control 800

4

.O0I

L - -

A A G W

1000 4 h - 800 I B Tau As

a00 aool 2004

L A W

I I I d- o 30 60 90 iao is0 300

Time ( 5 )

FIG. 9. Original recordings showing the glutamate-induced [Ca*+]i rise in single rat cerebellar granule cells. (A) Effect of 50 pM glutamate on [Ca2+]i in neurons pretreated with vehicle. (B) Effect of 50 pM glutamate on [Ca2+Ii in neurons pretreated with 25 pM tau antisense oligonucleotide. (C) Resting levels of [Ca2+ji in untreated neurons. Tau antisense oligonucleotide or vehicle was added to the cultures I h before the glutamate pulse. Traces are recordings from three representative neurons. G, glutamate addition; W, wash.

paradigm granule cells were allowed to differentiate for 10-12 days in culture, a period of time sufficient to permit the development of neurites and synaptic contacts. This in v i m differentiation may be responsible for the unusual simultaneous expression of both juvenile and mature tau isoforms.

In man, the juvenile isoform of tau mRNA has been found in adult brain in a cell-specific manner. Particularly, neurons expressing mRNAs encoding tau isoforms containing three repeats are present in pyramidal cell bodies throughout all layers of the cerebral cortex and in both hippocampal granule and pyramidal cells (Goedert et af., 1990, 1991b). The same authors also showed that, in contrast with granule cells, pyramidal cells in the hippocampus also express the mature tau isoform. Thus, only a limited number of neurons of the adult human brain are able to coexpress both juvenile and mature tau isoforms and these are among the most vulnerable cells in the nervous system, i.e. the hippocampal pyramidal cells. It is noteworthy that the tau sequence deriving from the juvenile isoform of tau mRNA has been found in PHF (Wischik et al., 1988; Kondo el al., 1988).

Tau antisense decreases glutamate neurotoxicity I61 1

The intermolecular interaction between the products of the two different tau isoforms has also been proposed to serve as the nidus for polymerization in paired helical filaments (Kosik et al., 1989; Wille et al., 1992). Thus, the inappropriate combinatorial expression of mature of juvenile tau is a common feature of cerebellar granule cells differentiated in culture and selective neurons in the adult brain which may undergo neurodegeneration.

We found that short-term exposure of primary cultures of cerebellar granule cells from the neonatal rat brain to high concentrations of glutamate resulted in a significant increase of immunoreactivity to tau. This effect was detectable 2 h after the glutamate pulse with two distinct anti-tau antibodies, TAU-2 and Alz-50. Since both TAU-2 and Alz-50 antibodies do not discriminate between the phosphorylated and non-phosphorylated forms of tau, it was unclear whether or not glutamate activates specific protein kinases in cerebellar granule cells to increase the phosphorylation state of tau proteins.

We were therefore interested in evaluating whether the increase in tau immunoreactivity detected in cerebellar granule cells after the glutamate pulse was a consequence of increased tau gene transcription. We found that glutamate increases, in a time- and concentration- dependent fashion, both mature and juvenile tau mRNA species. The effects of glutamate on tau immunoreactivity and mRNA levels might be causally and sequentially related. The correlation is suggested on the bases of similar dose-response and time-course curves. However, the possibility that, at least in part, the increase in tau immunoreactivity derived from post-transcriptional regulation of the tau gene cannot be ruled out. Studies in primary cultures of hippocampal neurons have shown that the increased tau immunostaining which occurred within 1 h of exposure to glutamate was not prevented by the protein synthesis inhibitor cycloheximide (Mattson, 1990). Thus, it has been proposed that the glutamate-induced increase of intracellular calcium may lead to phosphorylation of site(s) of tau protein. This phosphorylation may alter tau proteins to make them better recognized by specific tau antibodies, such as 5E2 and Alz-50. This interpretation is shared by Sautiere el al. (1992), who found that the amount of total tau proteins detected by Western analysis with anti-tau antibody was not significantly increased in rat cortical cells exposed to 5 pM glutamate for 30 min and tested 48-96 h later. We agree with this view only partially since our results obtained by Western analysis show that exposure of cerebellar granule cells to 100 pM glutamate for 15 min results in an increased amount of total tau protein, at least 2 h after the pulse. Considerable further work, including accurate dose- response and time-course analysis as well as extended immunocytochemistry with specific antibodies directed towards phosphorylated and non-phosphorylated sites, is required to clarify the relative contributions of post-translational modifications in the glutamate- induced increase of tau immunoreactivity. Nevertheless, we found that the glutamate-induced increase of tau immunoreactivity detectable by immunocytochemistry and Western blot analysis was prevented by exposing the cells to a tau antisense oligonucleotide (see below).

Doses of glutamate that do not induce neuronal death are able to increase both the tau mRNA level of immunoreactivity to tau protein. These findings suggest that the increased tau expression detectable after high doses of glutamate is not a general response to degenerative stimuli, and raise the question whether or not tau accumulation reflects an early state of degeneration. Alternatively, tau expression may be a sign of potential neuron vulnerability. In this line is the work by Al-Ghoul etal. (1989), who showed that tau immunoreactivity is a marker for neurons susceptible. to naturally occurring cell death during brain development, and that by Mattson (1990), who demonstrated that in a heterogeneous cell preparation (a primary culture of hippocampal neurons) glutamate induced degeneration only

in those neurons showing changes in tau immunoreactivity. Finally, high levels of transcripts encoding different isoforms of tau protein are found specifically in the bodies of the most vulnerable cell types of the brain, which are the pyramidal cells of the cerebral cortex and hippocampus (Goedert et al., 1991a).

There is an emerging consensus that glutamate, through the activation of specific glutamate receptor subtypes, activates a series of immediate early genes, triggering a long-lasting transcriptional programme which may result in the regulation of the expression of various proteins (Szekely et al., 1990; Memo et al., 1991a,b). Particularly, it has been previously established that stimulation of NMDA-selective glutamate receptors that are present in primary cultures of cerebellar granule cells results in the induction of a number of immediate early genes, including c-fos, c-jun, jun-B and zif/268 (Szekely et al., 1990). The protein products of these genes have been postulated to function as nuclear third messengers in coupling receptor stimulation to long-term phenotypic changes in neurons. It is tempting to speculate that the tau gene is one of the target genes that are regulated by these transcriptional factors. However, as pointed out above, the possibility that the increase of tau mRNA levels derives from post-transcriptional regulation of the tau gene, i.e. increased mRNA stability, cannot be underestimated. Nevertheless, the functional contribution of individual proteins in processing the glutamate signal to induce neuronal death is still unknown. We thus investigated the possible involvement of tau proteins in the neurotoxic process activated by glutamate using the oligonucleotide antisense strategy. We found that preincubation of cerebellar granule cells with a specific tau antisense oligonucleotide resulted in inhibition of the glutamate-induced tau immunoreactivity and tau protein level. Specificity of this effect was proved since pretreatment of the cells with the sense of oligonucleotide did not change the ability of glutamate to increase tau immunoreactivity. Functionally, the inhibition of tau neosynthesis by tau antisense oligonucleotide treatment resulted in a significant decrease of the sensitivity of the neurons to neurotoxic concentrations of glutamate. This protective effect was not due to an impairment of the ability of glutamate to increase the intracellular calcium concentration, suggesting that the sustained and abnormally high glutamate-induced increase of [Ca2+Ii is not always related to neuron death.

Central nervous system development, function, plasticity and per- haps degeneration depend on the coordinated transcriptional modifications of sets of genes encoding proteins that are relevant to particular neuronal functions. In vitro studies with morphologically homogen- eous populations of neuronal cells in primary culture provide experimental avenues for the elucidation of the regulatory mechanisms for genetic programmes that are operative in various aspects of neuronal life. The present data indicate that neosynthesis of the cytoskeleton- associated tau protein is a crucial step in the cascade of events promoted by glutamate leading to neurodegeneration. Indeed, the selective blockade of the glutamate-induced increase of tau mRNA processing reduced neuron sensitivity to the glutamate insult. This, regulation of tau synthesis might represent a common pattern by which glutamate may induce axonal maturation in developing neurons and neurodegeneration in selected, vulnerable, differentiated neurons. This view is supported by morphological observations in Alzheimer's brain of numerous contorted processes from cell bodies of NFT- bearing neurons and supernumerary basilar dendrites on hippocampal pyramidal cells (Kosik, 1990). All these phenomena may be part of an uncontrolled growth response to established neurons. Since the inhibition of tau synthesis does not completely prevent but only decreases neuronal sensitivity to the glutamate-induced cell death programme, it is possible to speculate that accumulation of tau


in response to glutamate represents a molecular risk factor for neurodegeneration which contributes, together with others, to lowering the safety margin of neurons to excitotoxin-induced injury.

Acknowledgements We thank P. Davies for kindly supplying Alz-50 antibodies, P. Liberini and M. Grilli for critically reviewing the manuscript, and I. Perez for helpful suggestions in Western analysis. We are also grateful to Sir Martin Roth, B. H. Anderton, D. Choi and E. Costa for their continual warm encouragement in pursuing this work. This work was financially supported by grants from Consiglio Nazionale delle Ricerche and from the Sandoz Foundation for Gerontological Research.

Abbreviations HEPES N-(2-hydroxyethyl)piperazine-N‘-(2-ethanensulphonic acid) NIT neurofibrillary tangle NMDA N-methyl-0-aspartate PCR polymerase chain reaction PIPES sodium piperazine-N,N’-bis-(2-ethane sulphonate)

References Al-Ghoul, W. M. and Miller, M. W. (1989) Transient expression of Alz-50

immunoreactivity in developing rat neocortex: a marker for naturally occurring neuronal death? Brain Res., 481, 361-367.

Amagada, P. V., Marzloff, K. and Hyman, B. T. (1992) Distribution of Alzheimer type pathologic changes in nondemented elderly individuals matches the pattern in Alzheimer’s disease. Neurology, 42, I68 1-1 688.

Balazs, R., Hack, N., Jorgensen, 0. S. and Cotman, C. W. (1989) N-methyl+- aspartate promotes the survival of cerebellar granule cells: pharmacological characterization. Neurosci. Lett., 101, 24 1-246.

Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N. H. (1988) Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem., 43, 1369-1374.

Brewer, G. J. and Cotman, C. W. (1989) NMDA receptor regulation of neuronal morphology in cultured hippocampal neurons. Neurosci. Lett., 99. 268-273.

Brion, J. P., Passareiro, H., Nunez, J. and Flament-Durand, J. (1985) Immunological determinants of tau proteins are present in neurofibrillary tangles of Alzheimer’s disease. Arch. Biol., 95, 229-235.

Caceres, A. and Kosik, K. S. (1990) Inhibition of neurite polarity by tau antisense oligonucleotides in primary cerebellar neurons. Nature. 343, 461463.

Celi, F. S., Zenilman, M. E. and Schuldiner, A. R. (1993) A rapid and versatile method to synthesize internal standards for competitive PCR. Nucleic Acids Res., 21, 1047.

Choi, D. W. (1988) Glutamate neurotoxicity and diseases of the nervous system. Neuron, 1, 623-634.

Ciardo, A. and Meldolesi, J. (1991) Regulation of intracellular calcium in cerebellar granule neurons: effects of depolarization and of glutamatergic and cholinergic stimulation. J . Neurochem.. 56, 184-191.

Connor, J. A. (1986) Digital imaging of free calcium changes and of spatial gradients in growing processes in single, mammalian central nervous system cells. Proc. Natl Acad. Sci. USA, 83, 6179-6183.

Eguchi, Y., Itoh, T. and Tomizawa, J.-I. (1991) Antisense RNA. Annu. Rev. Biochem., 60, 631452.

Flament, S., Delacourte, A. and Mann, D. M. A. (1990) Phosphorylation of tau proteins: a major event during the process of neurofibrillary degeneration. A comparative study between Alzheimer’s disease and Down’s syndrome. Brain Res., 516, 15-19.

Fohrman, E. B., de Erausquin, G., Costa, E. and Wojcik, W. J. (1993) Muscarinic m3 receptors and dynamics of intracellular Ca2+ cerebellar granule neurons. Eu,: J . Phannacol., Mol. Pharmacol. Sect., 245, 263-27 1.

Goedert, M. and Spillantini, M. G. (1990) Molecular neuropathology of Alzheimer’s disease: in situ hybridization studies. Cell. Mol. Neurobiol., 10, 159-174.

Goedert, M., Wischik, C., Crowther, R., Walker, J. and Klug, A. (1988) Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer’s disease: identification as the microtubule- associated protein tau. Proc. Narl Acad. Sci. USA, 85, 405 14055.

Goedert, M., Sisodia, S. S. and Price, D. L. (1991a) Neurofibrillary tangles and P-amyloid deposits in Alzheimer’s disease. Cur,: Opin. Neurobiol.. 1, 44147.

Goedert, M., Crowther, R. A. and Garner, C. C. (1991b) Molecular characterization of microtubule-associated proteins tau and MAP2. Trends Neurosci., 5, 193-199.

Greenberg. S. G. and Davies, P. (1990) A preparation of Alzheimer paired helical filaments that displays distinct tau proteins by polyacrylamide gel electrophoresis. Proc. Nut/ Acad. Sci. USA, 87, 5827-583 I.

Grundke-Iqbal, I., Iqbal, K., Turg, Y.-C., Quinlan. M., Wisniewski, H. M. and Binder. L. I. ( I 986) Abnormal phosphorylation of the microtubule-associated protein tau in Alzheimer cytoskeletal pathology. Proc. Nut/ Acad. Sci. USA, 83, 49134917.

Hanemaaijer, R. and Ginzburg. I . (1991) Involvement of mature tau isoforms in the stabilization of neurites in PC12 cells. J . Neurosci. Res.. 30, 163-171.

Harada, A,, Oguchi, K., Okabe, S., Kuno, J., Terada. S., Oshima, T., Sato- Yoshitake, R., Takei, Y., Noda. T. and Hirokawa, N. (1994) Altered microtubule organization in small-calibre axons of mice lacking tau protein. Nature. 369, 48849 1 .

Herzog, W. and Weber, K. ( 1978) Fractionation of brain microtubule-associated proteins. Eu,: J . Biochem.. 92, 1-8.

Ihara, Y., Nukina, N., Miura. R. and Ogawara, M. (1986) Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer’s disease. J . Biochem.. 99, 1807- I 8 10.

Jones, K. H. and Senft, J. A. (1985) An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. J. Hisrochem. Cytochem.. 33, 77-84.

Kanai, Y., Takemura. R., Oshima, T., Mori, H., Ihara, Y., Yanagisawa, M., Masaki, T. and Hirokawa, N. (1989) Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA. J . Cell Biol.. 109, 1 173-1 184.

Kidd. M. (1963) Paired helical filaments in electron microscopy of Alzheimer’s disease. Narure. 197, 192-193.

Kondo, J., Honda, T., Mori, H., Hamada, Y., Miura, R., Ogawara, M. and Ihara, 1. (1988) The carboxyl third of tau is tightly bound to paired helical filaments. Neuron, 1, 827-834.

Kosik, K. S. (1990) Tau proteins and Alzheimer’s disease. Cur,: Opin. Cell Biol.. 2, 101-104.

Kosik, K. S., Joachim, C. L. and Selkoe, D. J. (1986) Microtubule-associated protein tau is the major antigenic component of paired helical filaments in Alzheimer’s disease. Proc. Natl. Acad. Sci. USA. 83, 4044-4048.

Kosik, K. S., Orecchio, L. D., Bakalis, S. and Neve, R. (1989) Developmentally regulated expression of specific tau sequences. Neuron, 2 , 1389-1 397.

Lee, G., Cowan, N. and Kirschner, M. (1988) The primary structure and heterogeneity of tau protein from mouse brain. Science, 239, 285-288.

Lee, G. (1990) Tau protein: an update on structure and function. Cell Motil. Cytoskeleton, 15, 199-203.

Lipton, S. A. and Rosenberg, P. A. (1994) Excitatory amino acids as a final common pathway for neurologic disorders. New Engl. J. Med.. 330,613-622.

Liu, W.-K., Ksiezak-Reding, H. and Yen, S.-H. (1991) Abnormal tau proteins from Alzheimer’s disease brain. J. Biol. Chem.. 266, 21723-21727.

Loomis, P. A,, Howard, T. H., Castleberry, R. P. and Binder, L. 1. (1990) Identification of nuclear T isoforms in human neuroblastoma cells. Proc. Natl. Acad. Sci. USA, 87, 8422-8426.

Malgaroli, A,, Milani, D., Meldolesi, J. and Pozzan, T. (1987) Fura-2 measurement of cytosolic free Ca2+ In monolayers and suspensions of various types of animal cells. J. Cell Biol., 105, 2145-2155.

Mattson, M. P. (1990) Antigenic changes similar to those seen in neurofibrillary tangles are elicited by glutamate and Ca2+ influx in cultured hippocampal neurons. Neuron, 4, 105-1 17.

McDonald, J. W. and Johnston, M. V. (1990) Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res. Rev., 15, 41-70.

Memo, M., Bovolin, P., Costa, E. and Grayson, D. R. (1991a) Regulation of y-aminobutyric acid-A receptor subunit expression by activation of N- methyl-D-aspartate-selective glutamate receptor. Mol . Pharmacol.. 39. 599-603.

Memo, M., Bovolin, P., Costa, E. and Grayson, D. R. (1991b) Expression of y-aminobutyric acid-A receptor subunit mRNAs in neurons differentiating in culture. In Costa, E. and Joh, T. (eds), Neurotransmitrer Regulation of Gene Expression. Fidia Research Foundation Symposium Series, Vol. 7. Thieme Medical Publishers, New York, pp. 125-131.

Mize, R. R., Holdefer, R. N. and Nabors, L. B. (1988) Quantitative immunocytochemistry using an image analyzer. I. Hardware evaluation, image processing and data analysis. J. Neurosci. Merhods. 26, 1-24.

Tau antisense decreases glutamate neurotoxicity 161 3

Papasozomenos, S. C. and Binder, L. 1. (1987) Phosphorylation determines two distinct species of tau in the central nervous system. Cell Moril. Cytoskeleton, 8, 505-525.

Pizzi, M., Ribola, M., Valerio, A., Memo, M. and Spano, P. F. (1991) Various Ca” entry blockers prevent glutamate-induced neurotoxicity. Eu,: J. Pharmacol.. 209, 169-173.

Pizzi, M., Valerio. A,, Ribola, M., Spano. P. F. and Memo, M. (1993) A tau antisense oligonucleotide decreases neuron sensitivity to excitotoxic injury. NeuroReport. 4, 823-826.

Pizzi, M., Fallacara, C., Consolandi. 0.. Memo, M. and Spano, P. F. (1994a) a-Amino-3-hydroxy-5-methyI-4-isoxazolepropionate and kainate differentially affect neuronal cytoarchitecture of rat cerebellar granule cells. Neurosci. Lett., 166. 77-80.

Pizzi, M.. Valerio, A., Anighi, V., Belloni, M., Alberici, A,, Spano, P. F. and Memo, M. (l994b) Antisense strategy unravels tau proteins as molecular risk factors for glutamate-induced neurodegeneration. Cell. Mol. Neurohiol.,

Pomara, N., Singh, R., Deptula, D., Chou, J. C.-Y., Schwartz, M. B. and LeWitt, P. A. (1992) Glutamate and other CSF amino acids in Alzheimer’s disease. Am. J. Psychiotry, 149, 251-254.

Rashid. N. A. and Cambray-Deakin, M. A. (1992) N-methyl-D-aspartate effects on the growth, morphology and cytoskeleton of individual neurons in vitro. Dev. Brain Res., 67, 301-308.

Rothman, S. M. and Olney, J. W. (1988) Excitotoxicity and the NMDA receptors. Trends Neurosci.. 10, 299-302.

Sautiere, P.-E., Sindou, P., Couratier, P., Hugon, J., Wattez, A. and Delacourte, A. (1992) Tau antigenic changes induced by glutamate in rat primary

14, 569-578.

culture model: a biochemical approach. Neumsci. Lett., 140, 206210. Simon, R. P. (ed.) (1992) Excitatory Amino Acids. Fidia Research Foundation

Symposium Series, Vol. 9. Thieme Medical Publishers, New York. Sindou, P., Couratier, P., Barthe, D. and Hugon, J. (1992) A dose-dependent

increase of tau immunostaining is produced by glutamate toxicity in primary neuronal cultures. Brain Res.. 572, 242-246.

Szekely, A. M., Costa, E. and Grayson, D. R. (1990) Transcriptional program coordination by NMDA-sensitive glutamate receptor stimulation in primary culture of cerebellar neurons. Mol. Pharmacol., 38, 624-633.

Turker, R. P., Binder, L. I., Viereck, C., Hemmings, B. A. and Matus, A. (1988) Sequential appearance of high- and low-molecular weight forms of MAP2 in developing cerebellum. J . Neurosci., 8, 450345 15.

Wang, Y., Loomis, P. A., Zinkowsky, R. P. and Binder, L. I. (1993) A novel tau transcript in cultured neuroblastoma cells expressing nuclear tau. J . Cell

Wille, H., Drewes, G., Biernat, J., Mandelkow, E.-M. and Mandelkow, E. ( I 992) Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro. J. Cell Biol., 118,

Wischik, C. M., Novak, M., Thogersen, H. C. and Crowther, R. A. (1988) Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease. Proc. Natl Acad. Sci. USA, 85, 45064510.

Wisniewski, K., Jervis, G. A., Moretz, R. C. and Wisniewski, H. M. (1979) Alzheimer neurofibrillary tangles in diseases other than senile and presenile dementia. Ann. Neurol., 5, 288-294.

Wolozin, B. L., Pruchinicki, A., Dickson, D. W. and Davis, P. (1986) A neuronal antigen in the brain of Alzheimer’s patients. Science, 232.648450.

B i d . . 121, 257-267.

573-584.

Inhibition of Glutamate-induced Neurotoxicity by a Tau Antisense Oligonucleotide in Primary Culture...

Documents

Transcript of Inhibition of Glutamate-induced Neurotoxicity by a Tau Antisense Oligonucleotide in Primary Culture...