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Selective Phosphorylation of Adult Tau Isoforms in Mature Hippocampal Neurons Exposed to Fibrillar Aβ
MCN
Molecular and Cellular Neuroscience 9, 220–234 (1997) Article No. CN970615
Selective Phosphorylation of Adult Tau Isoforms in Mature Hippocampal Neurons Exposed to Fibrillar Ab Adriana Ferreira,1 Qun Lu, Lisa Orecchio, and Kenneth S. Kosik Center for Neurologic Diseases, Department of Medicine (Division of Neurology), Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
How senile plaques and neurofibrillary tangles are linked represents a major gap in our understanding of the pathophysiology of Alzheimer’s disease. We characterized a hippocampal neuronal culture system in which tau undergoes maturation in vivo; rat neurons maintained in culture for more than 3 weeks replicated the splicing and phosphorylation changes that tau undergoes upon maturation in situ. Using this model system, we induced an Alzheimer-like neuritic dystrophy following the application of fibrillar b-amyloid. The dystrophy consisted of focal distortions and swellings within the neurites and an altered phosphorylation of the adult tau isoforms. Fibrillar b-amyloid induced the concomitant activation of MAP kinase and GSK3 b. The aberrant activation of several signaling pathways may lead to the abnormal phosphorylation of tau and neuritic degeneration.
INTRODUCTION One of the key outstanding questions in the pathogenesis of Alzheimer’s disease (AD) is how the two hallmark lesions, senile plaques and neurofibrillary tangles, are linked. Senile plaques are composed of extracellular deposits of b-amyloid peptide (Ab) derived by proteolytic cleavage from the amyloid precursor protein. These plaques are often surrounded by dystrophic neurites which contain paired helical filaments (PHFs). The major component of PHFs is self-assembled hyperphosphorylated tau polymers. Tau, a heat-stable microtubule-associated protein, is encoded by a single gene (Neve et al., 1986) and is alternatively spliced to generate 1 To whom correspondence should be addressed at present address: Department of Cell and Molecular Biology and Institute for Neuroscience, Northwestern University, Searle Building Room 5-474, 320 East Superior Street, Chicago, IL 60611. Fax: (312) 503 7345. E-mail: [email protected].
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at least six isoforms in the adult brain. At the carboxy terminus tau contains a series of imperfect repeats that bind to microtubules (Lee et al., 1988). Both the expression and the phosphorylation of the different tau isoforms are developmentally regulated (Goedert et al., 1989 & 1993; Kosik et al., 1989; Bramblett et al., 1993; Brion et al., 1993; Arioki et al., 1993). For example, in the rat cerebral cortex, three alternatively spliced exons designated 2, 3, and 10 appear at approximately Postnatal Day 8 (Kosik et al., 1989). Although the functions of exons 2 and 3 are unknown, it is likely that exon 10 increases the affinity of tau for microtubules because this exon consists of an additional repeated unit capable of binding to microtubules. The expression of the adultspecific exons correlates with a decrease in tau phosphorylation (Brion et al., 1993; Arioki et al., 1993). Several studies suggested that phosphorylation may regulate the ability of tau to bind to microtubules, and hence regulate the dynamic properties of the microtubules (Drechsel et al., 1992; Bramblett et al., 1993). In vitro studies have shown that the extracellular signalregulated kinase (ERK, also known as MAP kinase) (Drewes et al., 1992), cdk5 (Bauman et al., 1992; Kobayashi et al., 1993; Paudel et al., 1993), cdc2 kinase (Ledesma et al., 1992; Vulliet et al., 1992), and glycogen synthase kinase 3 (Hanger et al., 1992; Mandelekow et al., 1992) are able to phosphorylate tau proteins. The protein phosphatases 1, 2A, and 2B can dephosphorylate tau in vitro (Goedert et al., 1992; Drewes et al., 1993; Harris et al., 1993). Recently, Ab toxicity models have been used to study the effects of the peptide on tau phosphorylation (Takashima et al., 1993; Busciglio et al., 1994). The addition of fibrillar Ab, but not the soluble form, resulted in an increase in the phosphorylation of tau accompanied by a decrease in its microtubule binding 1044-7431/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
FIG. 1. Pattern of tau expression in hippocampal neurons in culture. (A) Western blot analysis of heat-stable fractions prepared from hippocampal cultures reacted with a tau antibody (clone 5E2). The arrow indicates a molecular mass of 66 kDa. The samples were obtained from hippocampal cultures grown for 1 (B), 7 (C), 14 (D), 21 (E), 30 (F), and 45 (G) days in vitro (DIV). Neurons in B–G were fixed and stained with a tau antibody (clone 5E2). Bar, 20 µm.
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ability (Busciglio et al., 1994). The increased tau phosphorylation correlated with an increased tau protein kinase I activity (Takashima et al., 1993). However, these studies were done in cultures of cortical neurons that express only fetal tau isoforms. Because Alzheimer’s disease affects mature neurons, we developed a culture system that better reproduces the conditions observed in the mature animal. We showed that the addition of fibrillar Ab to these cultures resulted in the appearance of neuritic morphologies accompanied by increased phosphorylation of adult tau isoforms. In this setting the activity of both MAPK and GSK3 b increased.
RESULTS Mature Tau Expression in Cultured Hippocampal Neurons To determine whether neurons from the embryonic hippocampus can undergo splicing to the adult isoforms in culture, immunoblots of heat-stable fractions were performed on samples that had been growing from 1 to 45 days in culture. The initial stages of differentiation of dissociated hippocampal neurons in culture have been extensively characterized (Dotti et al., 1988; reviewed by Craig and Banker, 1993). Briefly, after 1 day in culture most of the cells have extended several minor processes and one axon. The minor processes begin their differentiation after 5 days in culture and by 7 days they have acquired dendritic characteristics. Synapse formation begins at about the same time. With development in culture the axonal and dendritic networks become more complex (Figs. 1B–1G) and the number of synaptic contacts increases (Fletcher et al.,
FIG. 2. Comigration of tau proteins obtained from adult rat brain and mature hippocampal cultures. Western blot analysis of heat-stable fractions prepared from adult rat brain (AB) and hippocampal cultures grown for 45 days in vitro (DIV) were reacted with a tau antibody (clone 5E2). Note the comigration of tau proteins in both samples.
FIG. 3. RT-PCR analysis of tau expression in hippocampal cultures. Reverse-transcribed DNA was amplified using primers flanking tau exons 2 and 3 (A) and tau exon 10 (B). FH, fetal hippocampus; AH, adult hippocampus; AB, adult brain; DIV, days in vitro; AP, adult plasmid. The numbers on the left indicate base pairs.
1991). From Day 7 to Day 30 after plating, the mature tau isoform pattern emerged. Until 7 days in culture two tau immunoreactive bands of 50 and 48 kDa were detected. Four tau immunoreactive bands were detected when heat-stable fractions were prepared from 21-day-old cultures. When hippocampal neurons were cultured for 30 days or longer at least 5 bands were immunolabeled with tau antibodies (Fig. 1A). These bands comigrated with adult rat tau (Fig. 2). To confirm the shift from a fetal to an adult pattern of tau expression during hippocampal development in culture, tau mRNA was qualitatively analyzed by reverse transcriptase-PCR (RT-PCR). We determined the time course of expression of exons 2, 3, and 10, which are expressed only in adult animals (Kosik et al., 1989). Reverse transcribed mRNA was amplified with primers flanking exons 2 and 3 at the N-terminal region and exon 10 at the C-terminal region. Exon 2 was first detected in neurons grown in culture for 14 days, and exons 2 and 3 were both expressed after 3 weeks in culture (Fig. 3A). Exon 10 was expressed after 14 days in culture (Fig. 3B).
Tau Phosphorylation State and the Expression of Putative Tau Kinases and Phosphatases in Culture To analyze the extent of tau phosphorylation in hippocampal neurons in culture, several antibodies directed against different phosphorylated epitopes were used. Figure 4A shows immunoblots of heat-stable
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FIG. 4. (A) Pattern of tau phosphorylation in hippocampal cultures. Western blot analysis of heat-stable fractions obtained from hippocampal cultures grown for different numbers of days in vitro (DIV) reacted with tau antibodies directed to phosphorylated epitopes (clones AT8 and PHF-1). (B) Developmental expression of kinases and phosphatases in hippocampal cultures.
fractions prepared from 1- to 30-day-old cultures that reacted with PHF-1 antibody (directed to Ser 396/Ser 404) or AT8 antibody (directed to Ser 199/Ser 202). We detected highly phosphorylated forms of tau up to 21 days in culture. After 3 weeks in culture, the tau isoforms expressed in hippocampal neurons lost most of their immunoreactivity with antibodies specific for phosphoisoforms (Fig. 4A). We next analyzed the pattern of expression of putative tau kinases and phosphatases over the course of
neuronal development in culture. Immunoblots of whole cell extracts obtained from hippocampal neurons grown in culture for 1 to 30 days were reacted with antibodies directed to cdc2, ERK 1 and 2, cdk 5 kinases, protein phosphatase 1 (PP1), protein phosphatase 2 A (PP2A), and calcineurin (PP2B). The low levels of cdc2 kinase detected in 1-day-old cultures further declined during the first week in culture and remained low over the remainder of the 30-day period studied (Fig. 4B). ERK1 and ERK2 kinase levels were initially low, increased as
224 the cells developed up to 21 days, and then decreased at 30 days in culture (Fig. 4B). The decrease in total levels of these kinases was confirmed by a dot immunobinding assay which showed a 20% decrease after 21 days in culture (Fig. 9). Cdk5 protein levels increased between Days 1 and 7 in culture and then remain static (Fig. 4B). PP2A and calcineurin levels also increased with development in culture and remained high throughout the time period analyzed in this study. On the other hand, the highest levels of PP1 were detected in 7-day-old cultures (Fig. 4B).
Effect of Ab Treatment on Neuronal Morphology and on the Adult Tau Isoforms To study the effect of Ab treatment on neurons expressing adult tau isoforms, soluble or fibrillar forms of the peptide were administered to hippocampal neurons after 30 days in culture at a final concentration of 20 µM. Ab fibrils were prepared as previously described (Busciglio et al., 1995) and confirmed by immunostaining using an Ab antibody. Fibrillar structures could be seen randomly distributed throughout the cultures. Some of them deposited directly on top of cell bodies while others were located along neuritic processes (Fig. 5). After incubation for 4 days in the presence of Ab, the neurons were fixed and their morphology was analyzed. A subset of neurons in cultures treated with fibrillar Ab showed progressive alterations in morphology. Affected neurites tended to follow a more tortuous course, and many also displayed swellings, particularly in more distal portions of the neurites. These swellings were highly immunoreactive to tau antibodies directed against phosphorylated isoforms (Fig. 6). Similar alterations of the neuronal morphology were observed in cultures grown in the presence of the Ab 25–35 (20 µM), a fragment thought to be responsible for the neurotoxicity (Yankner et al., 1990; Giovanelli et al., 1995; Pike et al., 1995). No morphological alterations were detected in cultures treated with soluble Ab (data not shown). To determine the ultrastructural changes induced by fibrillar Ab and the fine structure of the varicosities, the experimentally treated hippocampal neurons were visualized by EM. As shown in Fig. 7, regions devoid of microtubules and organelles were readily observed in treated cultures. In other areas, the microtubules were reduced in number and length compared to control cultures (Fig. 7). We next analyzed the effect of the addition of Ab on tau phosphorylation. Heat-stable fractions were prepared from control hippocampal cultures after 34 days and from identical cultures treated with soluble or fibrillar Ab from Day 30 through Day 34. These fractions were analyzed by immunoblot with antibodies directed
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against tau phosphoepitopes (i.e., AT8, PHF-1). A marked increase in immunoreactivity with these antibodies was detected in cultures treated with Ab fibrils (Fig. 8A). This increase in immunoreactivity was detected in higher molecular weight isoforms compared to the isoforms phosphorylated by fibrillar Ab in young cultures (Fig. 8B; see also Busciglio et al., 1995). No changes in tau phosphorylation were detected in cultures treated with the soluble Ab when compared to controls. The immunoreactivity for antibodies directed against tau phosphoepitopes increases with exposure time of the culture to the Ab fibrils as well as with the increase in the final concentration of the peptide (Figs. 8B and 8C). The increase in tau phosphorylation is associated with a decrease in the cytoskeletal-associated tau protein, in particular the higher molecular weight isoforms (Fig. 8D).
Ab Treatment Activated MAP Kinase and GSK3b Following exposure to fibrillar Ab, ERK-1 activity was determined in immunocomplexes using myelin basic protein as a substrate. A 65% induction in ERK-1 activity could be detected after a 24-h treatment of fibrillar Ab. A significant increase in the activity of ERK-1 was still apparent in cultures treated with fibrillar Ab for 4 days (Fig. 9). A similar sustained activation was demonstrated in an independent assay for MAP kinase activity in which immunoblots reacted with an anti-phosphotyrosine antibody. In cultures treated with fibrillar Ab, proteins that comigrated with ERK-1 and -2 showed a sustained increased immunoreactivity with the antiphosphotyrosine antibody (data not shown). We also sought to determine whether fibrillar Ab was able to activate GSK3b. Its activity was determined in immunocomplexes using a prephosphorylated GSK substrate. Using the same time points and conditions as above, there was a 25% increase in GSK3b activity detected after a 1-day exposure of cultured hippocampal neurons to fibrillar Ab (Fig. 9). The activity of this kinase also remained elevated after 4 days.
DISCUSSION Developmental Regulation of Tau Expression in Cultured Hippocampal Neurons Most of the detailed studies that characterize the development of hippocampal cultures focus on the early elaboration of neurites and the acquisition of dendritic and axonal identity. Here we have described continued developmental changes well beyond 7 days when the cultured neurons have clearly definable axons and
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FIG. 5. Thirty-day hippocampal cultures treated with fibrillar Ab for 4 days were fixed and stained with a b-peptide antibody (B, D, F). Fibrillar deposits were uniformly distributed; some of the deposits were located on top of the cell body (C and D) while others could be seen on top of the neuritic network (E and F). Bars, 10 µm.
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FIG. 6. Control (A and B) and fibrillar Ab-treated (C–F) 34 days in culture hippocampal neurons were stained with a tubulin antibody (A, C, D, and E). Note the abnormal appearance of the neuritic processes in C–E. Arrows point to a tortoise process in C and to swellings in D. Varicosities along these degenerating processes are immunoreactive to AT8 antibody (F) No immunoreactivity for this antibody was detected in control cultures (B). Bar, 20 µm.
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FIG. 7. Ultrastructural alterations induced by fibrillar Ab treatment in hippocampal cultures. Representative micrographs were taken from control (A and B) and fibrillar Ab (C and D) treated 34 days in vitro hippocampal neurons. Note the apparent decrease in the number of mitochondria and microtubules in fibrillar Ab-treated neurons. In some areas the microtubules are displace toward the periphery of the processes (D). Bar, 200 nm.
dendrites and have formed synaptic connections. At approximately 2 to 3 weeks after plating, hippocampal neurons achieve additional developmental milestones. There is a switch in tau expression from the fetal to the mature pattern, there is decreased tau phosphorylation,
and MAP kinase activity falls. The questions that emerge from these observations are (a) what triggers this late maturation step and (b) what is functionally achieved during this late maturation stage? Several studies, including those in which tau was
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FIG. 8. Effect of Ab treatment on tau phosphorylation. (A) Western blot analysis of heat-stable fractions prepared from 30 days in vitro cultures treated for 4 days with 20 µM soluble Ab (lane 1), fibrillar Ab (lane 2), Ab fragment 25–35 (lane 3), or PBS (lane 4) and reacted with AT8 and PHF-1 antibodies. Note the increase in immunoreactivity for these antibodies induced by Ab in a fibrillar form and by the Ab fragment 25–35. (B) Western blot analysis of heat-stable fractions obtained from 7 (lanes 1–3) and 30 (lanes 4–6) days in vitro hippocampal cultures treated with 20 µM fibrillar Ab for 4 days (lanes 1 and 4), 2 days (lanes 2 and 5), or 1 day (lanes 3 and 6) and reacted with AT8 antibody. Note that the tau bands immunoreactive to AT8 in old cultures treated with fibrillar Ab are of higher molecular weight as compared to those phosphorylated in 7-day cultures. (C) Dose response of fibrillar Ab treatment in 7- (lanes 1–3) and 30- (lanes 4–6) day cultures treated for 4 days with 0.2 µM (lanes 1 and 3); 2 µM (lanes 2 & 4), and 20 µM (lanes 3 and 6) fibrillar Ab and reacted with AT8 antibody. Note that different isoforms of tau are phosphorylated in older cultures as compared to cultures grown for 7 days in vitro. (D) Western blot analysis of cytoskeletal fractions obtained from control (lane 1) or fibrillar Ab (20 µM) for 4 days (lane 2) treated 30 days in vitro hippocampal neurons reacted with a tubulin and a tau antibody (clone 5E2). Note the decrease in tau bound to the cytoskeleton in fibrillar Ab treated cultures. Arrows indicate 66 kDa molecular weight.
suppressed in culture (Caceres & Kosik, 1990) and those in which tau was expressed in nonneural cells (Knops et al., 1991), concur that both fetal and mature tau are competent to induce process elongation. Neurite elongation is clearly independent of the expression of a mature isoform pattern, and indeed, the rapid phases of both axonal and dendritic elongation take place during the first week in culture (Dotti et al., 1998), when hippocampal neurons express only fetal tau isoforms. To what extent is tau splicing governed by external or internal signals? External signals could originate by cell–cell interactions through the establishment of synaptic con-
tacts. In situ, the switch in tau expression to the mature isoforms takes place at rat Postnatal Day 8–10 (Kosik et al., 1989), a time that corresponds to active synapse formation. However, the possibility that synapse formation triggers the switch in tau expression seems unlikely in view of the time course of synaptogenesis in hippocampal cultures. Synapses can be detected as early as 5 days in cultured neurons (Fletcher et al., 1991; Ferreira et al., 1995, and submitted for publication), well before adult tau isoforms are expressed. The functional correlate of these late developmental phenomena may be related to the stabilization of neurites once synaptic
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FIG. 9. Graphs showing MAP kinase (A and B) and GSK3 b (C and D) protein levels (A and C) and activity (B and D) in cultures treated with fibrillar Ab. The numbers are expressed as the percentage taken as 100% of the values obtained in 21-day in vitro cultures. The arrows indicate the time of addition of the fibrillar Ab.
connections are established. The use of culture systems in which synaptogenesis is delayed (Ferreira et al., 1995, and submitted for publication; Chin et al., 1995) may resolve this question. External signals could also be generated by the release of trophic factors from neurons
or glia to the culture medium. If these factors were responsible for the expression of adult tau isoforms, young neurons cultured in the presence of those factors should express adult tau isoforms. This is not the case: freshly plated hippocampal neurons in mature culture-
230 conditioned medium failed to accelerate the expression of adult tau isoforms (data not shown). A different hypothesis could explain tau splicing as a mechanism regulated by an intrinsic autonomous timekeeper in CNS neurons. Adult tau isoforms are expressed in situ at approximately the same time that they are expressed in culture, i.e., for E18 rats, 14 days in culture is approximately equivalent to Postnatal Day 10 when the developmental switch occurs (Kosik et al., 1989). The existence of such a mechanism regulating developmental events has been reported for the clonal differentiation of oligodendrocytes. Cultured oligodendrocyte precursor cells seem to have an internal clock that counts cell divisions stimulated by trophic factors. After a certain number of divisions, the precursor cells become unresponsive to growth factors, stop dividing, and differentiate (Temple & Raff, 1986). The maturation process that tau undergoes in these cultures includes changes in its phosphorylation state that resemble those observed in the intact organism (Matsuo et al., 1994). This change in tau phosphorylation temporally correlates with a decrease in both the protein levels and the activation state of MAP kinase. Whether any of the ERKs have a role in directly phosphorylating tau or indirectly regulating the tau phosphorylation state remains unknown. Mature tau isoforms with their four repeated microtubule-binding sequences are more effective in stabilizing microtubules (Goedert and Jakes, 1990). In addition, the state of phosphorylation of tau regulates its binding to microtubules both in vitro and in vivo (Drechsel et al., 1992; Bramblett et al., 1993). A decrease in tau phosphorylation enhances the affinity of tau for microtubules and consequently stabilizes the microtubules (Drechsel et al., 1992). Following the exploratory behavior involved in target identification and the synaptic pruning associated with establishing the correct target, the cytoskeleton may assume a more rigid configuration late in development. The expression of mature tau, as well as other cytoskeletal modifications such as increasing the amount of neurofilaments, contributes to stabilization.
Microtubule Dissolution and Dual Activation of MAP Kinase and GSK3 by Fibrillar Ab After 30 days in culture some processes extend for several millimeters and display extensive synaptic contacts. The microtubules required to maintain these processes must be highly stable. Fibrillar Ab induces a loss of microtubules, often in a patchy manner, suggesting that local toxic effects are responsible. The microtubule disruption is associated with distortions in the neurite course and swellings. Because microtubules
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serve as tracks for organelle transport, their disruption may result in a local transport blockade and consequently swelling. This mechanism is thought to underlie certain other classes of toxic neuropathies in which focal swellings occur (reviewed in Kosik and Selkoe, 1983). Both the activity of MAP kinase and its protein levels were increased by treatment of the cultures with fibrillar Ab treatment. Secreted b-APP activated MAP kinase and induced increased tau phosphorylation in PC12 cells (Greenberg et al., 1994); however, the activation of this kinase by secreted b-APP declined very rapidly and therefore resembled the effects of growth factors. Fibrillar Ab treatment resulted in a persistent increase in MAP kinase activity that remained evident even after 4 days of treatment. Furthermore, fibrillar Ab also increased the activity of GSK3 b, a kinase with multiple substrates. Other studies have suggested that GSK3 b plays a role in the regulation of tau phosphorylation (Hanger et al., 1992; Mandelkow et al., 1992; Medina et al., 1996) and in the generation of amyloid neurotoxicity (Takashima et al., 1993). Under normal conditions, the activation of MAP kinase by MAP kinase kinase results in the inactivation of GSK3 b (Eldar-Finkelman et al., 1995), probably via a pathway that involves multisite phosphorylation and activation of p90rsk by MAP kinase (Sturgill et al., 1988; Chung et al., 1991). The activation of p90rsk by MAP kinase leads to the inhibition of GSK3 b. Indeed in vitro studies suggest that p90rsk is the component of the MAP kinase pathway that inactivates GSK3 b (Sutherland and Cohen, 1994; Sutherland et al., 1993). Thus, the coordinate activation of MAP kinase and GSK3 b represents an aberrant activation pattern that may lead to abnormal phosphorylation of tau and neuritic degeneration. Surprisingly, the tau isoforms phosphorylated following treatment of 30-day-old hippocampal cultures with fibrillar Ab are not the same isoforms phosphorylated in 7-day-old cultures exposed to Ab. The fetal tau isoforms present in 7-day-old cultures undergo increased phosphorylation following treatment. Although the fetal isoforms are still present in 30-day-old cultures (see Figs. 1 and 3), they remain unchanged after the addition of Ab fibrils. This differential response to the same treatment could be explained if the substrates or the kinases activated by fibrillar Ab are localized to different subcellular compartments at different stages of development. Alternatively, different kinases might be activated at different times during development. For example, the reactivation of MAP kinase after it becomes quiescent at about 3 weeks in culture may have quite distinct effects in a more mature milieu. Regardless of the mechanism, these results emphasize the
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importance of studying neurons which express adult tau isoforms to elucidate the altered phosphorylation state of cells exposed to fibrillar Ab.
EXPERIMENTAL METHODS Preparation of Hippocampal Cultures Neuronal cultures were prepared from the hippocampi of Embryonic Day 18 rat embryos as previously described (Goslin and Banker, 1988). Briefly, embryos were removed and their hippocampi dissected and freed of meninges. The cells were dissociated by trypsinization (0.25% for 15 min at 37°C) followed by trituration with a fire-polished Pasteur pipette and plated onto poly-L-lysine-coated coverslips in MEM with 10% horse serum. After 4 h the coverslips were transferred to dishes containing an astroglial monolayer and maintained in MEM containing N2 supplements (Bottenstein and Sato, 1979) plus ovalbumin (0.1%) and sodium pyruvate (0.1 mM). For biochemical experiments, hippocampal neurons were plated at high density directly onto poly-L-lysine-coated culture dishes in MEM with 10% horse serum. After 4 h, the medium was changed for glial-conditioned MEM containing N2 supplements (Bottenstein and Sato, 1979) plus ovalbumin (0.1%) and sodium pyruvate (0.1 mM).
method of Lowry et al. (1951) as modified by Bensadoun and Weinstein (1976). Sodium dodecyl sulfate–polyacrylamide gels were run according to Laemmli (1970). Transfer of protein to Immobilon membranes (Millipore, Bedford, MA) and immunodetection were performed according to Towbin et al. (1979) as modified by Ferreira et al. (1989). The following antibodies were used: b-tubulin (clone tub2-1, 1:1000; Sigma, St. Louis, MO); tau (clone 5E2, 1:100, Kosik et al., 1988); dephosphorylated tau (clone tau-1, 1:100, Boerhinger and Mannheim); phosphorylated tau (clone AT8, 1:100, Biosource International, PHF-1, 1:5, a generous gift from Dr. Peter Davies, Albert Einstein College of Medicine); cdc2 kinase (1:500, UBI); anti-protein phosphatase 1 (1:500), 2A (1:500), and 2B (1:1000, all from UBI); anti-ERK1 (1:500, UBI); antiphosphotyrosine (clone 4G10, 1:500, UBI); and antiCDK5 (1:250) and anti-ERK2 (1:500, Santa Cruz Biotechnology). Secondary antibodies conjugated to alkaline phosphatase (1:5000, Promega) were used for detection of microtubular proteins. Secondary antibodies conjugated to HRP (1:5000, Promega) followed by enhanced chemiluminescence reagents (Amersham) were used for the detection of kinases and phosphatases. Dot immunobinding assay was performed using 125Ilabeled secondary antibodies as originally described by Jan et al. (1984) and modified by Ferreira et al. (1989).
Polymerase Chain Reaction Protein Determination, Electrophoresis, Immunoblotting, and Dot Immunobinding Assay Cultures were rinsed twice in warmed PBS, scraped into Laemmli buffer, homogenized in a boiling water bath for 5 min, and centrifuged at 33,000 rpm. The supernatant was removed and stored at 280°C until use. Cytoskeletal fractions were prepared as previously described (Ferreira et al., 1989). Briefly, cultures were rinsed in a microtubule stabilizing buffer (MTSB) for 2 min, extracted in MTSB plus 0.2% Triton X-100 for 4 min, and scraped into Laemmli buffer. To prepare heat-stable fractions the cells were scraped into MES/NaCl buffer (100 mM MES, 0.5 mM MgCl2, 1 M NaCl, 2 mM DTT) and immediately boiled for 5 min. Following centrifugation the supernatant was diluted with Laemmli sample buffer (Smith et al., 1995). All extraction buffers contained protease inhibitors (10 µg/ml leupeptin, 1 µg/ml pepstatin, 10 µg/ml aprotinin, and 0.5 mM PMSF) and phosphatase inhibitors (20 mM sodium pyrophosphate, 20 mM NaF, and 1 mM sodium orthovanadate). The protein concentration in whole cell extracts, cytoskeletal fractions, or heat-stable fractions was determined by the
To obtain total mRNA, hippocampal neurons cultured for 1, 7, 14, 21, or 30 days were washed with RNase free PBS and scraped into 300 µl/60-mm dish of RNAzol B (TEL-TEST, Inc). Total mRNA was extracted with RNAzol B and chloroform, precipitated with isopropanol, and then reverse transcribed using random hexamers and the Perkin–Elmer GeneAmp RNA PCR Kit (N808-0017). Ten microliters of the reverse transcription reaction was the substrate for a 50-µl PCR with the following two primer sets RT25 (sense, 58TGT CCT CGC CTC CTG TCG ATT ATC A 38), corresponding to nucleotides 239 to 25, and RT15 (antisense, 58CTG GGA TCC TGG TGG CAT TGG ATG TGC CTTT 38), corresponding to nucleotides 489 to 459, and RT13 (sense, 58TCC ACT GAG AAC CTG AAG CAC CAG38), corresponding to nucleotides 756 to 779, and RT12 (antisense, 58TCC ATG ATC AGT GAC GCC CCA GG38), corresponding to nucleotides 1328 to 1306 of the tau cDNA sequence (Kosik et al., 1989), to detect the expression of exon 2-3, and 10, respectively. Thirty cycles (1 min at 95°C, 1 min at 60°C, and 10 min at 72°C) were performed in a DNA Thermal Cycler (Perkin–Elmer Cetus). PCR products were separated by
232 electrophoresis and visualized by ethidium bromide staining.
Ab Treatments Synthetic Ab (1–40) obtained from Sigma Chemical Co. was dissolved in double-distilled H2O at 3 mg/ml and further diluted in PBS (1:1) and used immediately (soluble form) or incubated for 4 days at 37°C to preaggregate the peptide (Lorenzo and Yankner, 1994). Ab was added to the culture medium at a final concentration of 20 µM and the cells were grown in its presence for 4 days. In dose–response experiments, the Ab was added at final concentrations ranging from 0.2 to 20 µM. For time course experiments, the neurons were grown in the presence of Ab for 1 to 4 days. In some experiments synthetic Ab 25–35 (Sigma) was added to the culture medium at a final concentration of 20 µM.
Immunocytochemical Procedures Cultures were fixed for 20 min with 4% paraformaldehyde in PBS containing 0.12 M sucrose. They were then permeabilized in 0.3% Triton X-100 in PBS for 5 min and rinsed twice in PBS. The cells were preincubated in 10% BSA in PBS for 1 h at 37°C and exposed to the primary antibodies (diluted in 1% BSA in PBS) overnight at 4°C. Finally, the cultures were rinsed in PBS and incubated with secondary antibodies for 1 h at 37°C. In addition to the antibodies mentioned above, the following antibodies were used: anti-a-tubulin (clone DM1A) and polyclonal anti-tubulin (Sigma), anti-b-amyloid (1–40) from Sigma, anti-mouse IgG fluorescein-conjugated, and antirabbit IgG rhodamine-conjugated (Boehringer Mannheim).
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Kinase Assay Control and Ab-treated hippocampal cultures were washed twice in ice-cold PBS with 1 mM sodium orthovanadate and scraped into immunoprecipitation buffer (50 mM NaCl, 1 mM EDTA, 5 mM EGTA, 10 mM Tris–HCl, pH 7.6) with 0.2% Nonidet-P40, 0.1% deoxycholate, protease inhibitors, and phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 20 mM sodium fluoride). Lysates were centrifuged and normalized to 50–100 µg total protein and precipitated for 1 h at 0°C with anti-ERK1 or GSK 3 b antibodies as previously described (Greenberg et al., 1994; Wang et al., 1994). MAP kinase immunocomplexes were assayed for kinase activity for 30 min at 30°C in a mixture containing 15 µg myelin basic protein, 50 µM [g-32 P]ATP (µCi; 1 Ci 5 37 GBq), 10 mM MgCl2, 5 mM benzamidine, 1 mM dithiothreitol, and 30 mM Hepes (pH 7.2) in a final volume of 30 µl. GSK3b immunocomplexes were assayed for kinase activity for 30 min at 30°C in a mixture containing 15 µg phospho-glycogen synthase peptide substrate (UBI), 50 µM [g-32 P]ATP (µCi; 1 Ci 5 37 GBq), 10 mM MgCl2, 5 mM benzamidine, 1 mM dithiothreitol, and 30 mM Hepes (pH 7.2) in a final volume of 30 µl. Proteins were separated in a 15% polyacrylamide gel and autoradiograms were obtained by exposing X-ray films to immunoblots. The films were then analyzed with a PhosphorImager equipped with quantitation software (Molecular Dynamics).
ACKNOWLEDGMENTS We thank A. Ben-Itzhak for her technical help during the initial phases of this work. This work was funded by grants from NIH to K.S.K., by a Research Supplement for Underrepresented Minorities to A.F., by a grant from the Bayer Corp. to K.S.K. and by a pilot grant from the Massachusetts Alzheimer’s Disease Research Center to A.F.
Electron Microscopy After 34 days in culture, fibrillar Ab-treated and control hippocampal neurons were fixed in 3.5% glutaraldehyde in culture medium for 30 min at 37°C, rinsed in phosphate buffer (0.125 M), and reacted in 1% OsO4 in 0.125 M phosphate buffer for 1 h at 37°C in the dark. They were then rinsed, dehydrated in increasing concentrations of methanol followed by acetone, and embedded in Epon. After polymerization, the coverslips were peeled off and the cells punched out of the block and remounted. The cells were sectioned parallel to the glass coverslip coating the substrate. Thin sections were counterstained with uranyl acetate and lead citrate and examined using a Jeol 100 CX electron microscope.
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Molecular and Cellular Neuroscience 9, 220–234 (1997) Article No. CN970615
Selective Phosphorylation of Adult Tau Isoforms in Mature Hippocampal Neurons Exposed to Fibrillar Ab Adriana Ferreira,1 Qun Lu, Lisa Orecchio, and Kenneth S. Kosik Center for Neurologic Diseases, Department of Medicine (Division of Neurology), Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
How senile plaques and neurofibrillary tangles are linked represents a major gap in our understanding of the pathophysiology of Alzheimer’s disease. We characterized a hippocampal neuronal culture system in which tau undergoes maturation in vivo; rat neurons maintained in culture for more than 3 weeks replicated the splicing and phosphorylation changes that tau undergoes upon maturation in situ. Using this model system, we induced an Alzheimer-like neuritic dystrophy following the application of fibrillar b-amyloid. The dystrophy consisted of focal distortions and swellings within the neurites and an altered phosphorylation of the adult tau isoforms. Fibrillar b-amyloid induced the concomitant activation of MAP kinase and GSK3 b. The aberrant activation of several signaling pathways may lead to the abnormal phosphorylation of tau and neuritic degeneration.
INTRODUCTION One of the key outstanding questions in the pathogenesis of Alzheimer’s disease (AD) is how the two hallmark lesions, senile plaques and neurofibrillary tangles, are linked. Senile plaques are composed of extracellular deposits of b-amyloid peptide (Ab) derived by proteolytic cleavage from the amyloid precursor protein. These plaques are often surrounded by dystrophic neurites which contain paired helical filaments (PHFs). The major component of PHFs is self-assembled hyperphosphorylated tau polymers. Tau, a heat-stable microtubule-associated protein, is encoded by a single gene (Neve et al., 1986) and is alternatively spliced to generate 1 To whom correspondence should be addressed at present address: Department of Cell and Molecular Biology and Institute for Neuroscience, Northwestern University, Searle Building Room 5-474, 320 East Superior Street, Chicago, IL 60611. Fax: (312) 503 7345. E-mail: [email protected].
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at least six isoforms in the adult brain. At the carboxy terminus tau contains a series of imperfect repeats that bind to microtubules (Lee et al., 1988). Both the expression and the phosphorylation of the different tau isoforms are developmentally regulated (Goedert et al., 1989 & 1993; Kosik et al., 1989; Bramblett et al., 1993; Brion et al., 1993; Arioki et al., 1993). For example, in the rat cerebral cortex, three alternatively spliced exons designated 2, 3, and 10 appear at approximately Postnatal Day 8 (Kosik et al., 1989). Although the functions of exons 2 and 3 are unknown, it is likely that exon 10 increases the affinity of tau for microtubules because this exon consists of an additional repeated unit capable of binding to microtubules. The expression of the adultspecific exons correlates with a decrease in tau phosphorylation (Brion et al., 1993; Arioki et al., 1993). Several studies suggested that phosphorylation may regulate the ability of tau to bind to microtubules, and hence regulate the dynamic properties of the microtubules (Drechsel et al., 1992; Bramblett et al., 1993). In vitro studies have shown that the extracellular signalregulated kinase (ERK, also known as MAP kinase) (Drewes et al., 1992), cdk5 (Bauman et al., 1992; Kobayashi et al., 1993; Paudel et al., 1993), cdc2 kinase (Ledesma et al., 1992; Vulliet et al., 1992), and glycogen synthase kinase 3 (Hanger et al., 1992; Mandelekow et al., 1992) are able to phosphorylate tau proteins. The protein phosphatases 1, 2A, and 2B can dephosphorylate tau in vitro (Goedert et al., 1992; Drewes et al., 1993; Harris et al., 1993). Recently, Ab toxicity models have been used to study the effects of the peptide on tau phosphorylation (Takashima et al., 1993; Busciglio et al., 1994). The addition of fibrillar Ab, but not the soluble form, resulted in an increase in the phosphorylation of tau accompanied by a decrease in its microtubule binding 1044-7431/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
FIG. 1. Pattern of tau expression in hippocampal neurons in culture. (A) Western blot analysis of heat-stable fractions prepared from hippocampal cultures reacted with a tau antibody (clone 5E2). The arrow indicates a molecular mass of 66 kDa. The samples were obtained from hippocampal cultures grown for 1 (B), 7 (C), 14 (D), 21 (E), 30 (F), and 45 (G) days in vitro (DIV). Neurons in B–G were fixed and stained with a tau antibody (clone 5E2). Bar, 20 µm.
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ability (Busciglio et al., 1994). The increased tau phosphorylation correlated with an increased tau protein kinase I activity (Takashima et al., 1993). However, these studies were done in cultures of cortical neurons that express only fetal tau isoforms. Because Alzheimer’s disease affects mature neurons, we developed a culture system that better reproduces the conditions observed in the mature animal. We showed that the addition of fibrillar Ab to these cultures resulted in the appearance of neuritic morphologies accompanied by increased phosphorylation of adult tau isoforms. In this setting the activity of both MAPK and GSK3 b increased.
RESULTS Mature Tau Expression in Cultured Hippocampal Neurons To determine whether neurons from the embryonic hippocampus can undergo splicing to the adult isoforms in culture, immunoblots of heat-stable fractions were performed on samples that had been growing from 1 to 45 days in culture. The initial stages of differentiation of dissociated hippocampal neurons in culture have been extensively characterized (Dotti et al., 1988; reviewed by Craig and Banker, 1993). Briefly, after 1 day in culture most of the cells have extended several minor processes and one axon. The minor processes begin their differentiation after 5 days in culture and by 7 days they have acquired dendritic characteristics. Synapse formation begins at about the same time. With development in culture the axonal and dendritic networks become more complex (Figs. 1B–1G) and the number of synaptic contacts increases (Fletcher et al.,
FIG. 2. Comigration of tau proteins obtained from adult rat brain and mature hippocampal cultures. Western blot analysis of heat-stable fractions prepared from adult rat brain (AB) and hippocampal cultures grown for 45 days in vitro (DIV) were reacted with a tau antibody (clone 5E2). Note the comigration of tau proteins in both samples.
FIG. 3. RT-PCR analysis of tau expression in hippocampal cultures. Reverse-transcribed DNA was amplified using primers flanking tau exons 2 and 3 (A) and tau exon 10 (B). FH, fetal hippocampus; AH, adult hippocampus; AB, adult brain; DIV, days in vitro; AP, adult plasmid. The numbers on the left indicate base pairs.
1991). From Day 7 to Day 30 after plating, the mature tau isoform pattern emerged. Until 7 days in culture two tau immunoreactive bands of 50 and 48 kDa were detected. Four tau immunoreactive bands were detected when heat-stable fractions were prepared from 21-day-old cultures. When hippocampal neurons were cultured for 30 days or longer at least 5 bands were immunolabeled with tau antibodies (Fig. 1A). These bands comigrated with adult rat tau (Fig. 2). To confirm the shift from a fetal to an adult pattern of tau expression during hippocampal development in culture, tau mRNA was qualitatively analyzed by reverse transcriptase-PCR (RT-PCR). We determined the time course of expression of exons 2, 3, and 10, which are expressed only in adult animals (Kosik et al., 1989). Reverse transcribed mRNA was amplified with primers flanking exons 2 and 3 at the N-terminal region and exon 10 at the C-terminal region. Exon 2 was first detected in neurons grown in culture for 14 days, and exons 2 and 3 were both expressed after 3 weeks in culture (Fig. 3A). Exon 10 was expressed after 14 days in culture (Fig. 3B).
Tau Phosphorylation State and the Expression of Putative Tau Kinases and Phosphatases in Culture To analyze the extent of tau phosphorylation in hippocampal neurons in culture, several antibodies directed against different phosphorylated epitopes were used. Figure 4A shows immunoblots of heat-stable
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FIG. 4. (A) Pattern of tau phosphorylation in hippocampal cultures. Western blot analysis of heat-stable fractions obtained from hippocampal cultures grown for different numbers of days in vitro (DIV) reacted with tau antibodies directed to phosphorylated epitopes (clones AT8 and PHF-1). (B) Developmental expression of kinases and phosphatases in hippocampal cultures.
fractions prepared from 1- to 30-day-old cultures that reacted with PHF-1 antibody (directed to Ser 396/Ser 404) or AT8 antibody (directed to Ser 199/Ser 202). We detected highly phosphorylated forms of tau up to 21 days in culture. After 3 weeks in culture, the tau isoforms expressed in hippocampal neurons lost most of their immunoreactivity with antibodies specific for phosphoisoforms (Fig. 4A). We next analyzed the pattern of expression of putative tau kinases and phosphatases over the course of
neuronal development in culture. Immunoblots of whole cell extracts obtained from hippocampal neurons grown in culture for 1 to 30 days were reacted with antibodies directed to cdc2, ERK 1 and 2, cdk 5 kinases, protein phosphatase 1 (PP1), protein phosphatase 2 A (PP2A), and calcineurin (PP2B). The low levels of cdc2 kinase detected in 1-day-old cultures further declined during the first week in culture and remained low over the remainder of the 30-day period studied (Fig. 4B). ERK1 and ERK2 kinase levels were initially low, increased as
224 the cells developed up to 21 days, and then decreased at 30 days in culture (Fig. 4B). The decrease in total levels of these kinases was confirmed by a dot immunobinding assay which showed a 20% decrease after 21 days in culture (Fig. 9). Cdk5 protein levels increased between Days 1 and 7 in culture and then remain static (Fig. 4B). PP2A and calcineurin levels also increased with development in culture and remained high throughout the time period analyzed in this study. On the other hand, the highest levels of PP1 were detected in 7-day-old cultures (Fig. 4B).
Effect of Ab Treatment on Neuronal Morphology and on the Adult Tau Isoforms To study the effect of Ab treatment on neurons expressing adult tau isoforms, soluble or fibrillar forms of the peptide were administered to hippocampal neurons after 30 days in culture at a final concentration of 20 µM. Ab fibrils were prepared as previously described (Busciglio et al., 1995) and confirmed by immunostaining using an Ab antibody. Fibrillar structures could be seen randomly distributed throughout the cultures. Some of them deposited directly on top of cell bodies while others were located along neuritic processes (Fig. 5). After incubation for 4 days in the presence of Ab, the neurons were fixed and their morphology was analyzed. A subset of neurons in cultures treated with fibrillar Ab showed progressive alterations in morphology. Affected neurites tended to follow a more tortuous course, and many also displayed swellings, particularly in more distal portions of the neurites. These swellings were highly immunoreactive to tau antibodies directed against phosphorylated isoforms (Fig. 6). Similar alterations of the neuronal morphology were observed in cultures grown in the presence of the Ab 25–35 (20 µM), a fragment thought to be responsible for the neurotoxicity (Yankner et al., 1990; Giovanelli et al., 1995; Pike et al., 1995). No morphological alterations were detected in cultures treated with soluble Ab (data not shown). To determine the ultrastructural changes induced by fibrillar Ab and the fine structure of the varicosities, the experimentally treated hippocampal neurons were visualized by EM. As shown in Fig. 7, regions devoid of microtubules and organelles were readily observed in treated cultures. In other areas, the microtubules were reduced in number and length compared to control cultures (Fig. 7). We next analyzed the effect of the addition of Ab on tau phosphorylation. Heat-stable fractions were prepared from control hippocampal cultures after 34 days and from identical cultures treated with soluble or fibrillar Ab from Day 30 through Day 34. These fractions were analyzed by immunoblot with antibodies directed
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against tau phosphoepitopes (i.e., AT8, PHF-1). A marked increase in immunoreactivity with these antibodies was detected in cultures treated with Ab fibrils (Fig. 8A). This increase in immunoreactivity was detected in higher molecular weight isoforms compared to the isoforms phosphorylated by fibrillar Ab in young cultures (Fig. 8B; see also Busciglio et al., 1995). No changes in tau phosphorylation were detected in cultures treated with the soluble Ab when compared to controls. The immunoreactivity for antibodies directed against tau phosphoepitopes increases with exposure time of the culture to the Ab fibrils as well as with the increase in the final concentration of the peptide (Figs. 8B and 8C). The increase in tau phosphorylation is associated with a decrease in the cytoskeletal-associated tau protein, in particular the higher molecular weight isoforms (Fig. 8D).
Ab Treatment Activated MAP Kinase and GSK3b Following exposure to fibrillar Ab, ERK-1 activity was determined in immunocomplexes using myelin basic protein as a substrate. A 65% induction in ERK-1 activity could be detected after a 24-h treatment of fibrillar Ab. A significant increase in the activity of ERK-1 was still apparent in cultures treated with fibrillar Ab for 4 days (Fig. 9). A similar sustained activation was demonstrated in an independent assay for MAP kinase activity in which immunoblots reacted with an anti-phosphotyrosine antibody. In cultures treated with fibrillar Ab, proteins that comigrated with ERK-1 and -2 showed a sustained increased immunoreactivity with the antiphosphotyrosine antibody (data not shown). We also sought to determine whether fibrillar Ab was able to activate GSK3b. Its activity was determined in immunocomplexes using a prephosphorylated GSK substrate. Using the same time points and conditions as above, there was a 25% increase in GSK3b activity detected after a 1-day exposure of cultured hippocampal neurons to fibrillar Ab (Fig. 9). The activity of this kinase also remained elevated after 4 days.
DISCUSSION Developmental Regulation of Tau Expression in Cultured Hippocampal Neurons Most of the detailed studies that characterize the development of hippocampal cultures focus on the early elaboration of neurites and the acquisition of dendritic and axonal identity. Here we have described continued developmental changes well beyond 7 days when the cultured neurons have clearly definable axons and
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FIG. 5. Thirty-day hippocampal cultures treated with fibrillar Ab for 4 days were fixed and stained with a b-peptide antibody (B, D, F). Fibrillar deposits were uniformly distributed; some of the deposits were located on top of the cell body (C and D) while others could be seen on top of the neuritic network (E and F). Bars, 10 µm.
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FIG. 6. Control (A and B) and fibrillar Ab-treated (C–F) 34 days in culture hippocampal neurons were stained with a tubulin antibody (A, C, D, and E). Note the abnormal appearance of the neuritic processes in C–E. Arrows point to a tortoise process in C and to swellings in D. Varicosities along these degenerating processes are immunoreactive to AT8 antibody (F) No immunoreactivity for this antibody was detected in control cultures (B). Bar, 20 µm.
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FIG. 7. Ultrastructural alterations induced by fibrillar Ab treatment in hippocampal cultures. Representative micrographs were taken from control (A and B) and fibrillar Ab (C and D) treated 34 days in vitro hippocampal neurons. Note the apparent decrease in the number of mitochondria and microtubules in fibrillar Ab-treated neurons. In some areas the microtubules are displace toward the periphery of the processes (D). Bar, 200 nm.
dendrites and have formed synaptic connections. At approximately 2 to 3 weeks after plating, hippocampal neurons achieve additional developmental milestones. There is a switch in tau expression from the fetal to the mature pattern, there is decreased tau phosphorylation,
and MAP kinase activity falls. The questions that emerge from these observations are (a) what triggers this late maturation step and (b) what is functionally achieved during this late maturation stage? Several studies, including those in which tau was
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FIG. 8. Effect of Ab treatment on tau phosphorylation. (A) Western blot analysis of heat-stable fractions prepared from 30 days in vitro cultures treated for 4 days with 20 µM soluble Ab (lane 1), fibrillar Ab (lane 2), Ab fragment 25–35 (lane 3), or PBS (lane 4) and reacted with AT8 and PHF-1 antibodies. Note the increase in immunoreactivity for these antibodies induced by Ab in a fibrillar form and by the Ab fragment 25–35. (B) Western blot analysis of heat-stable fractions obtained from 7 (lanes 1–3) and 30 (lanes 4–6) days in vitro hippocampal cultures treated with 20 µM fibrillar Ab for 4 days (lanes 1 and 4), 2 days (lanes 2 and 5), or 1 day (lanes 3 and 6) and reacted with AT8 antibody. Note that the tau bands immunoreactive to AT8 in old cultures treated with fibrillar Ab are of higher molecular weight as compared to those phosphorylated in 7-day cultures. (C) Dose response of fibrillar Ab treatment in 7- (lanes 1–3) and 30- (lanes 4–6) day cultures treated for 4 days with 0.2 µM (lanes 1 and 3); 2 µM (lanes 2 & 4), and 20 µM (lanes 3 and 6) fibrillar Ab and reacted with AT8 antibody. Note that different isoforms of tau are phosphorylated in older cultures as compared to cultures grown for 7 days in vitro. (D) Western blot analysis of cytoskeletal fractions obtained from control (lane 1) or fibrillar Ab (20 µM) for 4 days (lane 2) treated 30 days in vitro hippocampal neurons reacted with a tubulin and a tau antibody (clone 5E2). Note the decrease in tau bound to the cytoskeleton in fibrillar Ab treated cultures. Arrows indicate 66 kDa molecular weight.
suppressed in culture (Caceres & Kosik, 1990) and those in which tau was expressed in nonneural cells (Knops et al., 1991), concur that both fetal and mature tau are competent to induce process elongation. Neurite elongation is clearly independent of the expression of a mature isoform pattern, and indeed, the rapid phases of both axonal and dendritic elongation take place during the first week in culture (Dotti et al., 1998), when hippocampal neurons express only fetal tau isoforms. To what extent is tau splicing governed by external or internal signals? External signals could originate by cell–cell interactions through the establishment of synaptic con-
tacts. In situ, the switch in tau expression to the mature isoforms takes place at rat Postnatal Day 8–10 (Kosik et al., 1989), a time that corresponds to active synapse formation. However, the possibility that synapse formation triggers the switch in tau expression seems unlikely in view of the time course of synaptogenesis in hippocampal cultures. Synapses can be detected as early as 5 days in cultured neurons (Fletcher et al., 1991; Ferreira et al., 1995, and submitted for publication), well before adult tau isoforms are expressed. The functional correlate of these late developmental phenomena may be related to the stabilization of neurites once synaptic
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FIG. 9. Graphs showing MAP kinase (A and B) and GSK3 b (C and D) protein levels (A and C) and activity (B and D) in cultures treated with fibrillar Ab. The numbers are expressed as the percentage taken as 100% of the values obtained in 21-day in vitro cultures. The arrows indicate the time of addition of the fibrillar Ab.
connections are established. The use of culture systems in which synaptogenesis is delayed (Ferreira et al., 1995, and submitted for publication; Chin et al., 1995) may resolve this question. External signals could also be generated by the release of trophic factors from neurons
or glia to the culture medium. If these factors were responsible for the expression of adult tau isoforms, young neurons cultured in the presence of those factors should express adult tau isoforms. This is not the case: freshly plated hippocampal neurons in mature culture-
230 conditioned medium failed to accelerate the expression of adult tau isoforms (data not shown). A different hypothesis could explain tau splicing as a mechanism regulated by an intrinsic autonomous timekeeper in CNS neurons. Adult tau isoforms are expressed in situ at approximately the same time that they are expressed in culture, i.e., for E18 rats, 14 days in culture is approximately equivalent to Postnatal Day 10 when the developmental switch occurs (Kosik et al., 1989). The existence of such a mechanism regulating developmental events has been reported for the clonal differentiation of oligodendrocytes. Cultured oligodendrocyte precursor cells seem to have an internal clock that counts cell divisions stimulated by trophic factors. After a certain number of divisions, the precursor cells become unresponsive to growth factors, stop dividing, and differentiate (Temple & Raff, 1986). The maturation process that tau undergoes in these cultures includes changes in its phosphorylation state that resemble those observed in the intact organism (Matsuo et al., 1994). This change in tau phosphorylation temporally correlates with a decrease in both the protein levels and the activation state of MAP kinase. Whether any of the ERKs have a role in directly phosphorylating tau or indirectly regulating the tau phosphorylation state remains unknown. Mature tau isoforms with their four repeated microtubule-binding sequences are more effective in stabilizing microtubules (Goedert and Jakes, 1990). In addition, the state of phosphorylation of tau regulates its binding to microtubules both in vitro and in vivo (Drechsel et al., 1992; Bramblett et al., 1993). A decrease in tau phosphorylation enhances the affinity of tau for microtubules and consequently stabilizes the microtubules (Drechsel et al., 1992). Following the exploratory behavior involved in target identification and the synaptic pruning associated with establishing the correct target, the cytoskeleton may assume a more rigid configuration late in development. The expression of mature tau, as well as other cytoskeletal modifications such as increasing the amount of neurofilaments, contributes to stabilization.
Microtubule Dissolution and Dual Activation of MAP Kinase and GSK3 by Fibrillar Ab After 30 days in culture some processes extend for several millimeters and display extensive synaptic contacts. The microtubules required to maintain these processes must be highly stable. Fibrillar Ab induces a loss of microtubules, often in a patchy manner, suggesting that local toxic effects are responsible. The microtubule disruption is associated with distortions in the neurite course and swellings. Because microtubules
Ferreira et al.
serve as tracks for organelle transport, their disruption may result in a local transport blockade and consequently swelling. This mechanism is thought to underlie certain other classes of toxic neuropathies in which focal swellings occur (reviewed in Kosik and Selkoe, 1983). Both the activity of MAP kinase and its protein levels were increased by treatment of the cultures with fibrillar Ab treatment. Secreted b-APP activated MAP kinase and induced increased tau phosphorylation in PC12 cells (Greenberg et al., 1994); however, the activation of this kinase by secreted b-APP declined very rapidly and therefore resembled the effects of growth factors. Fibrillar Ab treatment resulted in a persistent increase in MAP kinase activity that remained evident even after 4 days of treatment. Furthermore, fibrillar Ab also increased the activity of GSK3 b, a kinase with multiple substrates. Other studies have suggested that GSK3 b plays a role in the regulation of tau phosphorylation (Hanger et al., 1992; Mandelkow et al., 1992; Medina et al., 1996) and in the generation of amyloid neurotoxicity (Takashima et al., 1993). Under normal conditions, the activation of MAP kinase by MAP kinase kinase results in the inactivation of GSK3 b (Eldar-Finkelman et al., 1995), probably via a pathway that involves multisite phosphorylation and activation of p90rsk by MAP kinase (Sturgill et al., 1988; Chung et al., 1991). The activation of p90rsk by MAP kinase leads to the inhibition of GSK3 b. Indeed in vitro studies suggest that p90rsk is the component of the MAP kinase pathway that inactivates GSK3 b (Sutherland and Cohen, 1994; Sutherland et al., 1993). Thus, the coordinate activation of MAP kinase and GSK3 b represents an aberrant activation pattern that may lead to abnormal phosphorylation of tau and neuritic degeneration. Surprisingly, the tau isoforms phosphorylated following treatment of 30-day-old hippocampal cultures with fibrillar Ab are not the same isoforms phosphorylated in 7-day-old cultures exposed to Ab. The fetal tau isoforms present in 7-day-old cultures undergo increased phosphorylation following treatment. Although the fetal isoforms are still present in 30-day-old cultures (see Figs. 1 and 3), they remain unchanged after the addition of Ab fibrils. This differential response to the same treatment could be explained if the substrates or the kinases activated by fibrillar Ab are localized to different subcellular compartments at different stages of development. Alternatively, different kinases might be activated at different times during development. For example, the reactivation of MAP kinase after it becomes quiescent at about 3 weeks in culture may have quite distinct effects in a more mature milieu. Regardless of the mechanism, these results emphasize the
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Phosphorylation of Adult Tau Isoforms Induced by Ab
importance of studying neurons which express adult tau isoforms to elucidate the altered phosphorylation state of cells exposed to fibrillar Ab.
EXPERIMENTAL METHODS Preparation of Hippocampal Cultures Neuronal cultures were prepared from the hippocampi of Embryonic Day 18 rat embryos as previously described (Goslin and Banker, 1988). Briefly, embryos were removed and their hippocampi dissected and freed of meninges. The cells were dissociated by trypsinization (0.25% for 15 min at 37°C) followed by trituration with a fire-polished Pasteur pipette and plated onto poly-L-lysine-coated coverslips in MEM with 10% horse serum. After 4 h the coverslips were transferred to dishes containing an astroglial monolayer and maintained in MEM containing N2 supplements (Bottenstein and Sato, 1979) plus ovalbumin (0.1%) and sodium pyruvate (0.1 mM). For biochemical experiments, hippocampal neurons were plated at high density directly onto poly-L-lysine-coated culture dishes in MEM with 10% horse serum. After 4 h, the medium was changed for glial-conditioned MEM containing N2 supplements (Bottenstein and Sato, 1979) plus ovalbumin (0.1%) and sodium pyruvate (0.1 mM).
method of Lowry et al. (1951) as modified by Bensadoun and Weinstein (1976). Sodium dodecyl sulfate–polyacrylamide gels were run according to Laemmli (1970). Transfer of protein to Immobilon membranes (Millipore, Bedford, MA) and immunodetection were performed according to Towbin et al. (1979) as modified by Ferreira et al. (1989). The following antibodies were used: b-tubulin (clone tub2-1, 1:1000; Sigma, St. Louis, MO); tau (clone 5E2, 1:100, Kosik et al., 1988); dephosphorylated tau (clone tau-1, 1:100, Boerhinger and Mannheim); phosphorylated tau (clone AT8, 1:100, Biosource International, PHF-1, 1:5, a generous gift from Dr. Peter Davies, Albert Einstein College of Medicine); cdc2 kinase (1:500, UBI); anti-protein phosphatase 1 (1:500), 2A (1:500), and 2B (1:1000, all from UBI); anti-ERK1 (1:500, UBI); antiphosphotyrosine (clone 4G10, 1:500, UBI); and antiCDK5 (1:250) and anti-ERK2 (1:500, Santa Cruz Biotechnology). Secondary antibodies conjugated to alkaline phosphatase (1:5000, Promega) were used for detection of microtubular proteins. Secondary antibodies conjugated to HRP (1:5000, Promega) followed by enhanced chemiluminescence reagents (Amersham) were used for the detection of kinases and phosphatases. Dot immunobinding assay was performed using 125Ilabeled secondary antibodies as originally described by Jan et al. (1984) and modified by Ferreira et al. (1989).
Polymerase Chain Reaction Protein Determination, Electrophoresis, Immunoblotting, and Dot Immunobinding Assay Cultures were rinsed twice in warmed PBS, scraped into Laemmli buffer, homogenized in a boiling water bath for 5 min, and centrifuged at 33,000 rpm. The supernatant was removed and stored at 280°C until use. Cytoskeletal fractions were prepared as previously described (Ferreira et al., 1989). Briefly, cultures were rinsed in a microtubule stabilizing buffer (MTSB) for 2 min, extracted in MTSB plus 0.2% Triton X-100 for 4 min, and scraped into Laemmli buffer. To prepare heat-stable fractions the cells were scraped into MES/NaCl buffer (100 mM MES, 0.5 mM MgCl2, 1 M NaCl, 2 mM DTT) and immediately boiled for 5 min. Following centrifugation the supernatant was diluted with Laemmli sample buffer (Smith et al., 1995). All extraction buffers contained protease inhibitors (10 µg/ml leupeptin, 1 µg/ml pepstatin, 10 µg/ml aprotinin, and 0.5 mM PMSF) and phosphatase inhibitors (20 mM sodium pyrophosphate, 20 mM NaF, and 1 mM sodium orthovanadate). The protein concentration in whole cell extracts, cytoskeletal fractions, or heat-stable fractions was determined by the
To obtain total mRNA, hippocampal neurons cultured for 1, 7, 14, 21, or 30 days were washed with RNase free PBS and scraped into 300 µl/60-mm dish of RNAzol B (TEL-TEST, Inc). Total mRNA was extracted with RNAzol B and chloroform, precipitated with isopropanol, and then reverse transcribed using random hexamers and the Perkin–Elmer GeneAmp RNA PCR Kit (N808-0017). Ten microliters of the reverse transcription reaction was the substrate for a 50-µl PCR with the following two primer sets RT25 (sense, 58TGT CCT CGC CTC CTG TCG ATT ATC A 38), corresponding to nucleotides 239 to 25, and RT15 (antisense, 58CTG GGA TCC TGG TGG CAT TGG ATG TGC CTTT 38), corresponding to nucleotides 489 to 459, and RT13 (sense, 58TCC ACT GAG AAC CTG AAG CAC CAG38), corresponding to nucleotides 756 to 779, and RT12 (antisense, 58TCC ATG ATC AGT GAC GCC CCA GG38), corresponding to nucleotides 1328 to 1306 of the tau cDNA sequence (Kosik et al., 1989), to detect the expression of exon 2-3, and 10, respectively. Thirty cycles (1 min at 95°C, 1 min at 60°C, and 10 min at 72°C) were performed in a DNA Thermal Cycler (Perkin–Elmer Cetus). PCR products were separated by
232 electrophoresis and visualized by ethidium bromide staining.
Ab Treatments Synthetic Ab (1–40) obtained from Sigma Chemical Co. was dissolved in double-distilled H2O at 3 mg/ml and further diluted in PBS (1:1) and used immediately (soluble form) or incubated for 4 days at 37°C to preaggregate the peptide (Lorenzo and Yankner, 1994). Ab was added to the culture medium at a final concentration of 20 µM and the cells were grown in its presence for 4 days. In dose–response experiments, the Ab was added at final concentrations ranging from 0.2 to 20 µM. For time course experiments, the neurons were grown in the presence of Ab for 1 to 4 days. In some experiments synthetic Ab 25–35 (Sigma) was added to the culture medium at a final concentration of 20 µM.
Immunocytochemical Procedures Cultures were fixed for 20 min with 4% paraformaldehyde in PBS containing 0.12 M sucrose. They were then permeabilized in 0.3% Triton X-100 in PBS for 5 min and rinsed twice in PBS. The cells were preincubated in 10% BSA in PBS for 1 h at 37°C and exposed to the primary antibodies (diluted in 1% BSA in PBS) overnight at 4°C. Finally, the cultures were rinsed in PBS and incubated with secondary antibodies for 1 h at 37°C. In addition to the antibodies mentioned above, the following antibodies were used: anti-a-tubulin (clone DM1A) and polyclonal anti-tubulin (Sigma), anti-b-amyloid (1–40) from Sigma, anti-mouse IgG fluorescein-conjugated, and antirabbit IgG rhodamine-conjugated (Boehringer Mannheim).
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Kinase Assay Control and Ab-treated hippocampal cultures were washed twice in ice-cold PBS with 1 mM sodium orthovanadate and scraped into immunoprecipitation buffer (50 mM NaCl, 1 mM EDTA, 5 mM EGTA, 10 mM Tris–HCl, pH 7.6) with 0.2% Nonidet-P40, 0.1% deoxycholate, protease inhibitors, and phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 20 mM sodium fluoride). Lysates were centrifuged and normalized to 50–100 µg total protein and precipitated for 1 h at 0°C with anti-ERK1 or GSK 3 b antibodies as previously described (Greenberg et al., 1994; Wang et al., 1994). MAP kinase immunocomplexes were assayed for kinase activity for 30 min at 30°C in a mixture containing 15 µg myelin basic protein, 50 µM [g-32 P]ATP (µCi; 1 Ci 5 37 GBq), 10 mM MgCl2, 5 mM benzamidine, 1 mM dithiothreitol, and 30 mM Hepes (pH 7.2) in a final volume of 30 µl. GSK3b immunocomplexes were assayed for kinase activity for 30 min at 30°C in a mixture containing 15 µg phospho-glycogen synthase peptide substrate (UBI), 50 µM [g-32 P]ATP (µCi; 1 Ci 5 37 GBq), 10 mM MgCl2, 5 mM benzamidine, 1 mM dithiothreitol, and 30 mM Hepes (pH 7.2) in a final volume of 30 µl. Proteins were separated in a 15% polyacrylamide gel and autoradiograms were obtained by exposing X-ray films to immunoblots. The films were then analyzed with a PhosphorImager equipped with quantitation software (Molecular Dynamics).
ACKNOWLEDGMENTS We thank A. Ben-Itzhak for her technical help during the initial phases of this work. This work was funded by grants from NIH to K.S.K., by a Research Supplement for Underrepresented Minorities to A.F., by a grant from the Bayer Corp. to K.S.K. and by a pilot grant from the Massachusetts Alzheimer’s Disease Research Center to A.F.
Electron Microscopy After 34 days in culture, fibrillar Ab-treated and control hippocampal neurons were fixed in 3.5% glutaraldehyde in culture medium for 30 min at 37°C, rinsed in phosphate buffer (0.125 M), and reacted in 1% OsO4 in 0.125 M phosphate buffer for 1 h at 37°C in the dark. They were then rinsed, dehydrated in increasing concentrations of methanol followed by acetone, and embedded in Epon. After polymerization, the coverslips were peeled off and the cells punched out of the block and remounted. The cells were sectioned parallel to the glass coverslip coating the substrate. Thin sections were counterstained with uranyl acetate and lead citrate and examined using a Jeol 100 CX electron microscope.
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