Neuron,

Vol. 6, 445-454, March, 1991, Copyright

0 1991 by Cell Press

Neuronal Fodrin Proteolysis Occurs Independently of Excitatory Amino Acid-Induced Neurotoxicity Anna Maria M. Di Stasi,*+ Vittorio Callo,+*§ Marina Ceccarini,* and Tamara C. Petrucci* *Laboratory of Cell Biology *Laboratory of Physiopathology lstituto Superiore di Sanita ,Viale Regina Elena 299 Rome Italy

Summary In cultured cerebellar granule cells, the total amount of fodrin a subunit increased 3-fold between 0 and 10 days in vitro and fodrin mRNA increased 5-fold. The exposure of cerebellar neurons to NMDA induced the accumulation of a 150 kd proteolytic fragment of fodrin. The NMDA-induced breakdown of fodrin was time-, concentration-, and Ca*+dependent and was inhibited by APV, Mg*+, or the calpain I inhibitor N-acetyl-leu-leu-norleucinal. Kainate caused fodrin proteolysis through indirect activation of NMDA receptors. Quisqualate was ineffective. The NMDA-induced degradation of fodrin occurred under conditions that did not cause degeneration of cultured cerebellar neurons. These results show that Ca*+/ calpain I-dependent proteolysis of fodrin is selectively associated with NMDA receptor activation; however, fodrin proteolysis per se does not play a causal role in NMDA-induced toxicity in cerebellar granule cells. Introduction The membrane skeleton is a macromolecular assembly of structural proteins underlying the plasma membrane of eukaryotic cells. The major component of this structure is spectrin, a flexible rod-shaped molecule composed of two subunits, a and b (Goodman et al., 1988). In mammalian erythrocytes, spectrin crosslinks F-actin and associateswith several other proteins to form a submembrane meshwork (Marchesi, 1985) that is involved in the maintenance of cell shape, elasticity, and cell surface distribution (Marchesi, 1985; Coleman et al., 1989). Spectrin concentration is high in the brain, and several types of neurons have been shown to contain this cytoskeletal protein distributed over the cytoplasmic face of the entire neuronal plasma membrane (Bennett et al., 1982; Zagon et al., 1984, 1986). In the cerebellum, there is also evidence for a developmental regulation and a regional segregation of spectrin subtypes in different parts of a neuron (Riederer et al., 1987; Lazarides and Nelson, 1983), suggesting a role for compartmentalized spectrin sub+ The first two authors contributed equally to this report. 5 Present address: Unit on Molecular Neurobiology, Laboratory of Developmental Neurobiology, NICHD, National Institutes of Health, Bethesda, Maryland 20892.

types in the generation of neuronal plasma membrane diversity (Lazarides et al., 1984; see also Coleman et al., 1989, for a review). Fodrin (brain spectrin) shares several biochemical properties with other spectrin-like proteins (Coleman et al., 1989), is thought to be involved in organizing receptor and transmembrane channel domains (Bloch and Morrow, 1989; Srinivasan et al., 1988), and may participate in the control of vesicle traffic at the plasma membrane (Perrin et al., 1987). In vitro and in vivo studies have shown that fodrin can be rapidly cleaved by the ubiquitous Ca2+-activated neutral proteases or calpains (Baudry et al., 1981; Siman et al., 1984) and that the preferred location of calpain I cleavage is at a single site near the center of the a-fodrin (Harris et al., 1988). The proteolysis of fodrin may be physiologically significant: it is part of the biochemical mechanism by which Ca2+ stimulates secretion in chromaffin cells (Perrin et al., 1987), and it has been correlated with platelet activation (Samis et al., 1987) and neutrophil degranulation (Pontremoli et al., 1988). Calpain I activation induces the appearance of a proteolytic fragment(s) of the fodrin a subunit (150 kd) under conditions that favor the entry of Ca2+ through excitatory amino acid receptor-operated channels in the nerve cell membrane (Siman and Noszek, 1988; Seubert et al., 1988; Siman et al., 1989). Studies performed in therat hippocampus havesuggested,therefore, that the Ca*+-dependent proteolysis of fodrin can underlie phenomena such as long-term potentiation or may play an important role in the neurotoxic action of excitatory amino acids (Seubert et al., 1988; Siman et al., 1989). In the present study, we report the expression of fodrin during development in a homogeneous population of cultured cerebellar neurons, the granule cells, which are known to acquire in vitro several morphological (Thangnipon et al., 1983; Kingsbury et al., 1985; Gallo et al., 1986), biochemical (Gallo et al., 1982; Levi et al., 1984; Burgoyne et al., 1988), and electrophysiological (Hockberger et al., 1987; Cull-Candy et al., 1989) properties of differentiated nerve cells. A large number of studies have also demonstrated that cerebellar granule cells in culture express excitatory amino acid receptors and channels (Nicoletti et al., 1986; Novelli et al., 1987; Gallo et al., 1987a, 1990; CullCandy et al., 1988) and are susceptible to tehe toxic action of glutamate and its analogs (Favaron et al., 1988). We have, therefore, investigated whether glutamate receptor activation and subsequent opening of channels permeable to Ca*+ would cause calpain l-induced degradation of fodrin in cerebellar granule cells. Furthermore, we haveaddressed thequestion of whether the proteolytic cleavage of fodrin is directly responsible for the cell disintegration and death observed after exposure to excitatory amino acids (Favaron et al., 1988).

Figure 1. lmmunofluorescence

Micrographs

of Cultured

Cerebellar

Granule

Ceils Stained

with Anti-a-Fodrin

Antibodies

Cultures were fixed, permeabilized, and labeled with anti-a-fodrin affinity-purified antibodies followed by fluorescein-conjugated goat anti-rabbit immunoglobulin. (A and B) 3 DIV cultures. (C and D) 10 DIV cultures. (A) and (C) show the phase-contrast images of the fields shown in(B) and (D) (staining with anti-a-fodrin antibodies), respectivelv. Note that thecell bodies and the neurites of cerebellar neurons were brightly stained at both ages in culture. Bar, 50 pm.

Results localization and Developmental Regulation of Fodrin in Cultured Cerebellar Granule Cells Cerebellar primary cultures prepared from postnatal day8 (P8) rats are greatly enriched in granule neurons, which represent more than 95% of the total cells present (Gallo et al., 1982; Levi et al., 1984). When plated on a poly+-lysine substrate, cerebellar neurons begin to grow neurites shortly after plating (Thangnipon et al., 1983) and by 3 days in vitro (DIV) have extended a complex network of fibers (Figure IA). When cerebellar primary cultures were stained with anti-fodrin antibodies by indirect immunofluorescence, both cell bodies and neurites of granule neurons were brightly stained (Figures IB and ID). A ring of fluorescence could be observed in the cell bodies of cerebellar granule cells, indicating that fodrin was localized mainly in the cell membrane or in a closely associated structure. The fluorescence intensity increased with time in culture (Figure ID).

A relative quantitation of fodrin in cultured cerebellar granule cells at different days in vitro was performed by Western blot analysis after SDS-PAGE of total cellular proteins. Figure 2A shows that the affinity-purified polyclonal antibody used selectively recognized a240 kd a subunit of fodrin in cultured cerebellar neurons: a gradual and continuous increase in the total amount of fodrin was observed after 3 DIV (Figure 2A). A scanning of three different autoradiograms showed that the total amount of fodrin (relative to total cellular proteins) increased by3-fold between 0 and 10 DIV (3 experiments). We also determined fodrin mRNA levels in cultured cerebellar granule cells and in cerebellar tissue at different stages of development. In cultured cerebellar neurons, a-fodrin 6.8 kb mRNA increased about 5-fold between 0 and IO DIV (Figure 2B). Similar results were observed in cerebellar tissue between P8 and P40 (data not shown). Conversely, levels of @actin and its mRNA declined in cultured cerebellar granule cells between 3 and IO DIV (data not shown).

Fodrin Regulation

in Cultured

Central Neurons

447

6

A

28s

A

-

18s -

123456 1*2

3

-4

10

123 Figure2. Granule

Expression of a-Fodrin and Its mRNAduring Cell Development in Culture

- B

Cerebellar

(A) Western blot analysis of total cellular proteins. Proteins were separated by SDS-PAGE on a 6% gel and analyzed by immunoblot techniques using an affinity-purified anti-a-fodrin antibody Lane 1: 0 DIV cultures. Lane 2: 3 DIV cultures. Lane 3: 5 DIV cultures. Lane 4: 10 DIV cultures. Equal amounts of total proteins (200 ug) were loaded in each lane. (B) Northern blot analysis of total RNA. The Northern blot was hybridized with a 3*P-labeled a-fodrin rat cDNA probe. Lane 1: 0 DIV cultures. Lane 2: 3 DIV cultures. Lane 3: 10 DIV cultures. Equal amounts of RNA (15 ug) were loaded in each lane. To compare the relative amounts of RNA loaded on the gel for each sample, a Polaroid 55 film negative of the photograph of the ethidium bromide-stained ribosomal bands on the blotted MY bond N filter was scanned by densitometry.

-0

100

200

NMDA

Fodrin Degradation Occurs via N-MethybAspartate Receptor Activation The set of developmental experiments described above indicates that a-fodrin expression in cerebellar neurons increases with time in culture, reaching a peak at 10 DIV. We decided, therefore, to study the effects of excitatory amino acid agonists on fodrin proteolysis in cerebellar cells cultured for 10 days. At this age, cerebellar granule cells have been shown to express excitatory amino acid receptors of the N-methyl-n-aspartate (NMDA), kainate, and quisqualate type (Nicoletti et al., 1986; Novelli et al., 1987; Gallo et al., 1987a, 1990; Cull-Candy et al., 1988). Exposure of cultured cerebellar neurons to either NMDA or kainate induced fodrin degradation. The a subunit of fodrin and its smaller proteolytic fragment(s) (150 kd) were detected by immunoblotting: in cultured cerebellar neurons exposed to NMDA or kainate at concentrations exceeding 20 PM, the amount of the 150 kd fragment(s) increased severalfold (Figure 3A). When both agonists were used at a concentration of 100 KM, NMDA produced a consistent increase in the 150 kd fragment(s), whereas kainate was much less effective. In a film exposed to the same blot, but for a shorter period of time, we observed adecrease in the intensity of the 240 kd band in the cultures treated with NMDA,

Figure NMDA

3. Induction in Cultured

300

400

pM

of a-Fodrin Proteolysis Cerebellar Ceurons

by Kainate

and

(A) Concentration dependence of NMDA and kainate effects. Cerebellar granule cells (10 DIV) were exposed to micromolar concentrations of excitatory amino acid receptor agonists for 40 min. Total proteins were separated by SDS-PAGE and analyzed by immunoblot techniques. Lane 1: control. Lane 2: 20 uM kainate. Lane 3: 100 uM kainate. Lane 4: :20 uM NMDA + 5 PM glycine, Mg*+-free. Lane 5: 100 uM NMDA + 5 uM glycine, Mg2+free. Lane 6: 100 uM quisqualate. The molecular mass values in kilodaltons for a-fodrin and its proteolytic fragment(s) are indicated on the left. (EL)Dose-response of NMDA-induced a-fodrin proteolysis. Cerebellar neurons (IO DIV) were incubated for 40 min with increasing concentrations of NMDA (in the absence of MgZ’ and in the presence or absence of 5 pM glycine) and immediately harvested for SDS-PAGE and immunoblot analysis. Relative quantitation of the 150 kd proteolytic fragment(s) was performed by scanning densitometry of the autoradiograms. Closed squares: NMDA + glycine. Open square: NMDA without glycine.

but not in those treated with kainate. The scanning of such an autoradiogram gave the following values for the 240 kd band (in arbitrary units): control = 15.5; NMDA = 11.5; kainate = 15.3. The values for the 150 kd band were as follows: control = 0; NMCIA = 1; kainate = 0. The effect of kainate was counteracted bythe NMDA receptor antagonist DL-2-aminod-phos-

Nl?UrO” 448

240

-

123456 Figure 4. Time Course in Cultured Cerebellar

of NMDA Neurons

Effects on a-Fodrin

Proteolysis

Cerebellar granule cells (IO DIV) were incubated with 100 PM NMDA + 5 PM glycine in the absence of Mg*’ for different periods of time and immediately harvested for SDS-PAGE and immunoblot analysis. Lane 1: control. Lane 2: 5 min incubation. Lane 3: 20 min incubation. Lane 4: 40 min incubation. Lane 5: 80 min incubation. Lane 6: 120 min incubation.

phonovaleric acid (APV) (data not shown), suggesting that an indirect activation of NMDA receptors was involved in the effects of kainate (see Results and Discussion). Finally, the other glutamate receptor agonist, quisqualate, did not cause any degradation of fodrin (Figure 3A). A dose-response curve for NMDA-induced accumulation of the 150 kd fragment(s) is shown in Figure 38. The effect of NMDA in the absence of extracellular Mg2’ and in the presence of 5 PM glycine increased linearly with the agonist concentration up to 100 uM and reached a plateau at a concentration between 100 and 300 PM. When glycine was omitted from the

extracellular medium, the effects of 100 uM NMDA were reduced to control levels (Figure 3B, open square). The effect of NMDA on fodrin degradation was time dependent. Figure 4 shows that, when cultured cerebellar neurons were exposed to 100 uM NMDA for progressively increasing periods of time, the relative proportion of the 150 kd proteolytic fragment(s) increased up to 80 min and then consistently decreased (3 experiments). The NMDA-induced degradation of fodrin was totally inhibited by the NMDA receptor antagonist APV (100 PM) or by the addition of 3 mM external Mg*+(Figure 5A). The effect of NMDA was also prevented by the omission of Ca2+ in the extracellular medium or by the addition of the membrane-permeable calpain I inhibitor N-acetyl-Leu-Leunorleucinal (Figure 5A; Tsujinata et al., 1988). Interestingly, the membrane-impermeable calpain I inhibitor leupeptin was ineffective in modifying NMDA-induced fodrin breakdown (Figure 5A). Depolarization of 10 DIV cerebellar neurons with 50 mM KCI did not induce any increase in the 150 kd fragment(s) of fodrin (Figure 5B). When cerebellar neurons were cultured only for 3 days and then exposed to kainate or NMDA (100 PM), no fodrin degradation was observed (Figure 5B). Parallel 4sCa2+experiments showed that both NMDA and kainate stimulated 45Ca2+influx into cerebellar granule cells cultured for 10 days, but they were both ineffective in 3 DIV cultures (Table 1). Similar results were previously obtained with high extracellular K+ concentration (Gallo et al., 1987b). Effect of NMDA Receptor Activation on Calpain I Breakdown To gain direct evidence for calpain I activation during NMDA-induced proteolysis of a-fodrin, we analyzed Figure 5. NMDA Effects on a-Fodrin Proteolysis in Mature and immature Cerebellar Neurons in Culture

(A) Modulation of NMDA-induced a-fodrin proteolysis. Cerebellar granule cells (10 DIV) were incubated for 40 min with 100 240uM NMDA under various experimental 240conditions and immediately harvested for SDS-PAGE and immunoblot analysis. Lane 1: control. Lane 2: 100 PM NMDA + 5 PM 150glycine, Mg*‘-free. Lane 3: 100 uM NMDA + 5 uM glycine + 100 PM APV, Mgz’-free. Lane 4: 100 uM NMDA + 5 PM glycine, Mg2’- and Ca2+-free. Lane 5: 100 uM NMDA + 5 PM glycine + 3 m M Mg2+. Lane 6: 100 uM NMDA + 5 uM glycine + 10 ug/ml calpain I inhibitor (N-acetyl-Leu-Leu-norleucinal), Mg2+1234567 free. Lane 7: 100 uM NMDA + 5 PM glycine + IO ug/ml leupeptin, Mg2+-free. The molecular mass values in kilodaltons for a-fodrin and its proteolytic fragment(s) are indicated on the left. (6) Absence of NMDA and kainate effects on a-fodrin proteolysis in immature cerebellar neurons and absence of high K’ effects on a-fodrin proteolysis in mature cerebellar neurons. Cerebellar granule cells (3 DIV: lanes l-3; IO DIV: lane 4) were incubated with either NMDA, kainate, or a 50 m M KCCcontaining Krebs’ Ringer medium for 40 min. Cells were immediately harvested for SDS-PAGE and immunoblot analysis. Lane 1: control. Lane 2: 100 PM kainate. Lane 3: 100 PM NMDA + 5 PM glycine, Mg*+-free. Lane 4: 50 m M KCI Ringer solution.

12

34

Fodrin Regulation 449

in Cultured

Central Neurons

Table 1. NMDA- and Kainate-Stimulated Cultured Cerebellar Neurons

Wa*+

Uptake

into

45Ca2c Uptake (% of Control) Days In Vitro

NMDA

Kainate

3 10

120 f 15 230 + 10

105 k 8 194 * 9

Cells were washed in Krebs’ Ringer medium, preincubated at 37OC for 10 min, and then incubated for 5 min in fresh Krebs’ Ringer medium (Mg*+-containing or Mgzf-free) containing 1 uCi/ ml 45Ca*+ in the presence or absence of 100 PM NMDA + 5 uM glycine or 100 uM kainate. Results are the average * SEM of 3 experiments: in each experiment, 3 dishes of cells exposed to NMDA or kainate were compared with 3 dishes of control. 45Ca2+ uptake in controls was 4.5 it 0.7 nmol per mg of protein per 5 min. __

whether autoproteolysis of the enzyme (Croall, 1989; Harris and Morrow, 1988) would occur in cultured cerebellar neurons after stimulation with the glutamate agonist. Western blot analysis performed by using polyclonal antibodies to the catalytic (84 kd) and the regulatory (26 kd) subunits of bovine heart calpain I revealed that activation of the enzyme, after exposure of the cultured neurons to NMDA, caused a reduction in the amount of its regulatory subunit, accompanied by an increase in the amount of a 17 kd fragment (Figure 6). NMDA-Induced Fodrin Degradation and Its Relationship to Toxicity In a separate set of experiments, cerebellar granule cells were exposed to 100 PM NMDA or kainate for 40 min in Krebs’ Ringer medium at 37Y, returned to their “self-conditioned” culture medium, and examined 24 hr later for their viability. The ionic conditions chosen to study excitotoxicity (Mg*+-containing medium for kainate and Mg2+-free medium for NMDA) were identical to those used in the experiments on fodrin degradation, to allow a direct comparison between these two types of experiments. Observation of the cultures by phase-contrast microscopy showed that cerebellar neurons exposed to NMDA were still completely intact after 24 hr, whereas neurons exposed to kainate showed clear signs of toxicity: their cell bodies were phase-dark and pyknotic, and their network of fibers had degenerated. These results were confirmed by fluorescein diacetate-propidium iodide double staining of the cultures (Jones and Senft, 1985). Figure 7 shows that after a 40 min exposure to NMDA, no significant increase in the percentage of dead cells was observed, compared with controls (17% + 3% dead cells in control cultures, 19% k 2% dead cells in NMDA-treated cultures; n = 24fields per culture). After exposure to kainate, 65% f 3% of the total cells stained with propidium iodide (n = 24 fields per culture, P < 0.001 by two-tailed t-test). Kainate toxicity was only slightly attenuated by APV. In 2 separate experiments, only 5% and 14% fewer cells died when APV (100 PM) was added to kainate (100 ).tM) in the medium, compared with kainate alone.

1234567 Figure 6. Autoproteolysis bellar Neurons

of Calpain

I in NMDA-Stimulated

Cere

Cultured cerebellar granule cells (IO DIV) were incubated with kainateor NMDAfor40 min and immediately harvested for SDSPAGE and immunoblot analysis. Bound anti-a-fodrin antibody was detected with alkaline phosphatase-conjugated goat antibody to rabbit immunoglobulin. Lane 1: P8 rat cerebellar homogenate. Lane 2: rat erythrocyte ghosts. Lane 3: cultured cerebellar neurons, control. Lane 4: cultured cerebellar neurons, 20 PM kainate. Lane 5: cultured cerebellar neurons, 100 uM kainate. Lane 6: cultured cerebellar neurons, 20 uM NMDA + 5 uM glytine, Mg2+-free. Lane 7: cultured cerebellar neurons, 100 uM NMDA + 5 uM glycine, Mgzc-free. The arrowheads on the right indicate (from the top) the catalytic (84 kd) and the regulatory (26 kd) subunits of calpain I and the major proteolytic product of the enzyme (17 kd).

100 1 4 a

0 u 2

80 1

60

0 40 z zg? 20 n i,l Control Figure7. Neurotoxicity NMDA or Kainate

100 FM NMDA

Assay in Cerebellar

100 PM Kainate Neurons

Exposed to

CuIturedcerebellargranulecells~10DIV~wereincubatedat37”C with NMDA (100 uM) + glycine (5 PM) or kainate (100 FM) in a Mg2+-free (NMDA)or in a Mg*+-containing (kainate) Krebs’ Ringer medium for 40 min. Cells were returned to their self-conditioned culture medium at 37°C for 24 hr and then stained with fluorescein diacetate-propidium iodide. Controls were incubated either in a Mgzf-free or in a Mg*+-containing medium for 40 min, with no agonists. The omission of Mg’+ did not, by itself, cause any significant cell death. The percentage of dead cells in controls was the same in the presence or absence of Mg2+. Results are expressed as a percentage of total cells counted. Bars indicate SEM.

Discussion We have studied brane cytoskeleton consisting of intact rat cerebellum and

a major component of the memof nerve cells, fodrin, in a system central neurons isolated from the cultured in vitro. The primary cul-

Nel.lKSl 450

tures used in the present study contain more than 95% granule cells, the most abundant neuronal cell population present in the rat postnatal cerebellar cortex (Altman, 1972a). This in vitro system has allowed us to study the expression of fodrin during neuronal development without the interference of other neural cell types that also contain this protein (Zagon et al., 1984; Riederer et al., 1987). Since cerebellar granule neurons express several cytoskeletal (Burgoyne and Cambray-Deakin, 1988; Vitadiello and Donis-Donini, 1990) and neuron-specific proteins (Gallo et al., 1986) during their development in culture, we have first analyzed the pattern of expression of fodrin in these cells at different times in vitro. Western and Northern blot experiments showed that both fodrin and its mRNAs were present in cultured granule neurons almost immediately after plating (Figure 2), indicating that fodrin is already expressed in granule cells actively dividing in the external granular layer of the cerebellar cortex (Altman, 1972a). Levels of fodrin and its mRNAs increased during the first 5 days in culture, i.e., during the period of maximal neurite outgrowth and morphological differentiation of the cerebellar neurons (Gallo et al., 1982; Thangnipon et al., 1983). At 3 DIV, both the cell bodies and the extensive neurite network of granule neurons were brightly stained with anti-fodrin antibodies (Figure 1). In a second phase of development in vitro (between 5 and 10 DIV), cerebellar granule cells acquire several properties of differentiated neurons, including the expression of voltage-operated ionic channels (Kingsbury and Balazs, 1987; Carboni and Wojcik, 1988; Cull-Candy et al., 1989) and of receptors for the two major neurotransmitters of the cerebellar cortex, glutamate and y-aminobutyric acid (Gallo et al., 1985, 1986,199O; Vicini et al., 1986; Nicoletti et al., 1986; Novelli et al., 1987; Cull-Candy et al., 1988). The acquisition of these functional properties indicates that the maturation of cerebellar granule neurons in vitro occurs on schedule, since in vivo approximately 3 weeks following birth are necessary to establish completely functional synaptic contacts between granule cells and mossy fibers and between granule and Purkinje cells (Woodward et al., 1971; Altman, 1972b; Puro and Woodward, 1977; Crepe1 et al., 1982, J. Physiol., abstract). Levels of a-fodrin and its mRNA also increased between 5 and 10 DIV (Figures 1 and 2), in agreement with previous immunocytochemical studies in vivo (Riederer et al., 1987; Siman et al., 1987) which showed that fodrin immunoreactivity was low in the developing internal granular layer, but high in granule neurons in the adult cerebellum. Experiments in progress in our laboratory show that both fodrin and its mRNAs increase severalfold during postnatal cerebellar development (data not shown). Our results indicate, therefore, that the pattern of expression of fodrin in granule neurons in vitro resembles the developmental changes observed in vivo. Several studies have shown that, in the mature brain, fodrin can be biochemically modified by the

action of proteolytic enzymes (Siman et al., 1984,1989; Goodman et al., 1988; Siman and Noszek, 1988). In particular, in the rat hippocampus, the activation of excitatory amino acid receptors of the NMDA and kainate subtype and the subsequent transmembrane Ca2+ influx have been related to calpain l-induced proteolysis of fodrin (Siman et al., 1984,1989; Seubert et al., 1988). However, a direct causal link between Ca*+ influx induced by excitatory amino acids, activation of Ca*+-dependent proteolytic enzymes (calpains), and fodrin cleavage has not been conclusively demonstrated in simple and homogeneous neuronal systems. We have studied the link between excitatory amino acid receptor activation and fodrin modifications in cultured cerebellar granule neurons, since these cells express all three subtypes of excitatory amino acid receptors (Nicoletti et al., 1986; Novelli et al., 1987; Cull-Candy et al., 1988; Gallo et al., 1987a, 1990) and areeasilyaccessibleto pharmacological manipulation at a stage of their development in vitro corresponding to maturegranule neurons (seediscussion above). We have addressed the following questions: First, which subtype(s excitatoryaminoacid receptors is linked to fodrin proteolysis in neurons? Second, is calpain I directly responsible for fodrin degradation? Finally, are the same excitatory amino acid receptors linked to calpain I activation and fodrin proteolysis? Our experiments show that Ca2+-stimulated, calpain I-dependent fodrin proteolysis is selectively associated with the functional activation of NMDA receptors. The effect of NMDA was concentration dependent in the micromolar range (Figure 3B) and was strongly inhibited by the selective antagonist APV (Figure 5A; Evans et al., 1982). Furthermore, NMDA caused fodrin degradation only in the presence of glycine (Figure 3B), which is a necessary coagonist to produce full activation of NMDA receptors (Johnson and Ascher, 1987; Mayer et al., 1989). The ionic dependence of the effect of NMDA on fodrin degradation was also consistent with the idea that NMDA-activated channels were directly involved. Extracellular Ca2+ was necessary to trigger the effect of NMDA, whereas the presence of Mg2+ in the extracellular medium totally inhibited the effects of the glutamate agonist (Figure 5A). Since NMDA-activated channels in neurons are mainly permeable to Ca2+ (MacDermott et al., 1986; Mayer and Westbrook, 1987) and are blocked by Mg2+ (Mayer et al., 1984; Nowak et al., 1984), the simplest explanation of these results is that a rapid influx of Ca2+ through channels of the NMDA receptor complex initiates the cascade of events that terminates with the proteolysis of fodrin. The rapid changes in intracellular Ca2+ caused by the activation of NMDA receptors and opening of Ca2+-permeable channels (MacDermott et al., 1986; Murphyet al., 1987) are in the rangeof activation of calpain I (Murachi et al., 1983). Furthermore, the calpain I inhibitor N-ace@-Leu-Leu-norleucinal, which can permeate the cell membrane (Tsujinata et al., 1988), strongly inhibited NMDA-induced degrada-

;odrin 151

Regulation

in Cultured

Central Neurons

tion of a-fodrin (Figure 5A). Finally, autoproteolysis of rhe regulatory subunit of calpain I was observed under stimulation with NMDA (Figure 6). Incubation of cultured cerebellar neurons with hother excitatory amino acid receptor agonists indicated that the effects of NMDA on fodrin proteolysis were selective. Quisqualate, which causes membrane depolarization in cultured cerebellar granule cells (Cull-Candy et al., 1988), induced only a modest increase in %a*+ uptake (Gallo et al., submitted) and did not induce any degradation of fodrin (Figure 3A). Kainate consistently stimulated “Ca*+ uptake (Table 1; see also Wroblewski et al., 1985), but caused only a small increase in the 150 kd proteolytic fragment(s) of fodrin, as measured by Western blot analysis (Figure 3A). The effect produced by kainate was, however, totally reversed by the NMDA antagonist APV (data not shown), showing that endogenous glutamate released from granule cells upon exposure to kainate (Patrizio et al., 1989) indirectly activated NMDA receptors, causing partial degradation of fodrin. In deed, kainate was the most potent excitatory amino acid receptor agonist causing endogenous glutamate release from cultured cerebellar neurons, whereas NMDA was ineffective (Levi et al., 1991). Kainateinduced cleavage of fodrin was also inhibited by the calpain I inhibitor N-acetyl-Leu-Leu-norleucinal (data not shown), indicating that the indirect effect of kainate through NMDA receptors occurred primarily via calpain I. Finally, depolarization of the cerebellar neurons with a high extracellular K+ concentration, which stimulates Ca*+ influx through voltage-dependent Ca*+ channels (Gallo et al., 1987b; Kingsbury and Balazs, 1987), did not causefodrin proteolysis (Figure5B). These results indicate that membrane depolarization and Ca*+ influx are not sufficient, by themselves, to cause proteolytic cleavage of fodrin. NMDA receptor activation and subsequent elevation of the intracellular Ca*+ concentration are directly involved in excitotoxicity in the central nervous system (see Choi, 1988, for a review). In the rat hippocampus, NMDA receptor activation in the presence of extracellular Ca*+ is required for excitatory amino acid-induced neurotoxicity (Nadler et al., 1981), and calpain I activation has been related to the damage of CA3 pyramidal cells (Siman et al., 1989). Cerebellar granule cells have been shown to be sensitive to the toxic action of glutamate and related compounds (Garthwaite and Garthwaite, 1986; Favaron et al., 1988). Under the experimental conditions adopted in the present study, however, NMDA did not cause degeneration of cultured cerebellar granule cells, whereas kainate did (Figure 7). On the other hand, NMDA caused substantial Ca*+/calpain l-induced proteolysis of a-fodrin, whereas kainate induced little or no direct calpain activation and subsequent fodrin proteolysis (see discussion above). Therefore, substantial Ca*+/ calpain l-induced proteolysis of a-fodrin occurs in cells exposed to NMDA under conditions that do not result in cerebellar granule cell degeneration, indicat-

ing that fodrin proteolysis is uncoupled from neurotoxicity. As proposed by Seubert et al. (1988) and by Siman et al. (1989), Ca2+ influx and calpain I activation may initiate excitatory amino acid-mediated neurotoxicity, whereas other regulatory processes are involved in the complete destruction of the linkage between the cell membrane and cytoskeleton. Harris and co-workers have recently reported that, in vitro, calmodulin regulates Ca*+/calpain l-dependent fodrin cleavage (Harris et al., 1989; Harris and Morrow, 1990). Interestingly, cleavage of the fodrin a subunit alone does not destroy the tetrameric form of fodrin and its binding to actin, whereas proteolysis of the a and p subunits (the latter being totally calmodulin dependent) rendersfodrin incapableof reforming tetramers and of binding actin (Harris and Morrow, 1990). On the basis of these results, it can be concluded that many Ca*+-triggered intracellular events regulate the interaction of fodrin with actin and, as a consequence, the physical interaction between the cell membrane and the cytoskeleton. In this context, it can be speculated that Ca*+/calpain l-dependent proteolytic cleavage of the a subunit alone may push a nerve cell to a “sensitive” or “pre-toxic” state that is, however, reversible. We are currently investigating the relationship between excitatory amino acid-induced neurotoxicity and the biochemical modifications of the cell membrane skeleton afterthe degradation of the fodrin a subunit. In conclusion, the expression of fodrin in cultured central neurons follows their general pattern of biochemical and functional maturation in vitro. We have demonstrated that fodrin can be posttranslationally modified by physiological stimuli occurring at the NMDA receptor-channel complex. Because of the selectivity of the NMDAeffects, it can be speculated that a small percentage or an isoform of fodrin is physically associated with the NMDA receptor complex in the plasma membrane. This pool of fodrin would be readily accessible to the Ca*+-dependent proteolytic degradation produced by calpain I. The cleavage of fodrin occurs also in the absence of NMDA-mediated neurotoxic effects, which indicates that fodrin proteolysis is neither necessary nor sufficient to mediate excitotoxicity. Experimental

Procedures

Cell Cultures Cerebellar neuronal cultures enriched in granule cells were prepared from P8 rats as previously described (Levi et al., 1984; Kingsbury et al., 1985). Cells were plated on 60 m m or 35 m m diameter dishes precoated with poly-t-lysine (5 @ml; M, 53,000) and cultured in Eagle’s basal medium containing 10% fetal calf serum, 25 m M KCI (final concentration), 2 m M glutamine, and 100 @ml gentamycin. Cell density was 3 x W/cm~. For immunocytochemical staining, cell suspensions were plated on poly-tlysine-coated, 12 m m round coverslips placed in 35 m m diameter dishes. Arabinosylcytosine (IO PM) was added to the cultures after 16-20 hr in vitro to inhibit the replication of nonneuronal cells.Thecultures were maintained in an atmosphereof 5% COz, 95% air and were used after 10 DIV, unless otherwise stated. The cultures were characterized immunocytochemically as pre-

Neuron 452

viously described (Aloisi et al., 1985; Kingsbury >95% of the cells were granule neurons.

et al., 1985), and

lmmunocytochemistry Fodrin was visualized by indirect immunofluorescence. Cells on coverslips were fixed for 15 min in 2% paraformaldehyde, permeabilized for 5 min with acetone at room temperature, and incubated for 30 min with anti-a-fodrin antibodies (1:lOO; see below). After several washes in PBS, the cells were incubated with goat anti-rabbit fluorescein-conjugated IgG (I:40 in PBS) for 30 min. Coverslipswerefinally mounted in PBS-glycerol (1:l) and examined using a Polyvar (Reichert) ultramicroscope equipped with interference contrast and fluorescein optics. Electrophoresis and lmmunoblots Cell cultures were directly solubilized in 4x SDS-PAGE buffer (Laemmli, 1970), collected after scraping with a rubber policeman, and heated at 90°C for 5 min. After multiple passages through a 21 gauge hypodermic needle, the samples were run on discontinuous 6% polyacrylamide-SDS slab gels (Laemmli, 1970). Gels (100 x 60 mm) were cast in a Hoefer electrophoresis apparatus. Each lane contained 200 pg of protein. Protein concentration was determined by a BCA (modified Lowry) assay (Pierce), using serum albumin as a standard. Proteins were transferred electrophoretically to nitrocellulose paper (Schleicher & Schuell, Inc., Keene, NH) (Towbin et al., 1979). After a 14 hr incubation in Tris-buffered saline solution VSS) containing 5% nonfat dry milk, blots were washed and incubated with anti-a-fodrin affinity-purified antibodies (1:lOO) in TBS-3% bovine serum albumin. After 1 hr, blots were washed and subsequently incubated with ‘251-labeled protein A for 1 hr. Blots were extensively washed in TBS plus 0.05% Tween-20 and finally dried. Autoradiography was performed using Kodak X-Omat AR5 film for 24-48 hr at -70°C with Cronex intensifying screens (DuPont). Relative levels of a-fodrin breakdown products were quantified by using a laser densitometer (Ultroscan XL, LKB) connected to an IBM computer. lmmunodetection by alkaline phosphatase was performed by using IgG-alkaline phosphatase-conjugated secondary antibodies and 4-chloro-I-naphthol or NTB/BClP as the substrate (Blake et al., 1984). RNA Isolation and Northern Blot Analysis Total RNA was prepared from cell cultures at different days in vitro by the guanidine isothiocyanate-cesium chloride method (Chirgwin et al., 1979). Total RNA (15 pg) was electrophoresed through a 1% agarose-formaldehyde denaturing gel (Sambrook et al., 1989), transferred to a nylon membrane, Hybond N (Amersham International, UK), and hybridized with a 2.5 kb a-fodrinselective cDNA (obtained from a rat brain library; kindly provided by Dr. Thomas L. Leto) high specific activity probe (>I@ cpm per pg of DNA). Hybridizations were performed at 42’C in 50% formamide, followed by two washes at 50°C and 60°C in 1 x SSC, 0.2% SDS. Incubation with Excitatory Amino Acid Receptor Agonists Culture dishes were washed twice with Krebs’ Ringer medium (128 m M NaCI, 5 m M KCI, 2.7 m M CaCl,, 1.2 m M MgSO+ 1 m M NazHP04, 10 m M glucose, 20 m M HEPES [pH 7.41) and incubated with excitatory amino acid receptor agonists (in the presence or absence of various antagonists/inhibitors) dissolved in the same medium. Alterations in the ionic composition of the medium are indicated in the figures. After incubation at 37OC for different times, the cultures were directly solubilized in 4x SDS-PAGE sample buffer and processed for electrophoresis. 45CaZ+ Uptake Cultures were washed twice with Krebs’ Ringer medium and preincubated in the same medium at 37OC. After 10 min, the medium was replaced with new Krebs’ Ringer medium containirig 1 pCi/ml ‘Ya*+ (20 Ci/mmol) and 100 FM kainate or 100 PM NMDA. After 5 min at 37OC, the cultures were rapidlywashed 3 times with 154 m M choline, 2 m M EDTA at room temperature and finally solubilized in 1 ml of 0.1 M NaOH. Different aliquots

of the NaOH extracts were used either for scintillation or for protein estimation (Lowry, 1951).

counting

Neurotoxicity Assay Cells were grown on poly-t-lysine-coated coverslips and used at 10 DIV. The culture medium was replaced with Krebs’ Ringer medium (128 m M NaCI, 5 m M KCI,2.7mM CaCI,, 2 m M Na2HPOI, 10 m M glucose, 20 m M HEPES, 5 PM glycine for incubation with NMDA; 128 m M NaCI, 5 m M KCI, 2.7 m M CaC&, 1.2 m M MgS04, 1 m M NazHP04, 10 m M glucose, 20 m M HEPES for incubation with kainate), and cells were incubated at 37OC for 10 min prior to the addition of NMDA or kainate. After a 40 min incubation with the glutamate agonists, cells were returned to their selfconditioned culture medium at 37°C and kept in a 95% air, 5% CO? atmosphere at 37OC for 24 hr. Cells were then assessed for toxicity by staining with fluorescein diacetate (IO wg/ml) and propidium iodide (Jones and Senft, 1985). Two coverslips from each culture dish were examined by fluorescence microscopy. Three to four representative fields from each coverslip were scored, and 80-IOOtotal cells per field werecounted. Viablecells appeared bright fluorescent green, whereas nonviable neurons were stained in red. Materials Fodrin was purified from bovine brain according to Harris et al. (1985). For antibody preparation, fodrin was injected into New Zealand rabbits using Freund’s adjuvant. Antibodies to fodrin were purified from immune sera by affinity chromatography on Sepharose 48 CNBr-activated resin (Pharmacia) coupled with bovine fodrin. Antibody titer was determined by ELISA. Bovine heart anti-calpain I antisera were generously provided by Dr. Dorothy E. Croall. Y-labeled protein A and 45CaC12 were from Amersham International (Little Chalfont, England), calpain inhibitor I (N-acetyl-Leu-Leu-norleucinal) was from Boehringer Mannheim (Germany), and leupeptin was from Sigma Chemical Co. (St. Louis, MO). Goat anti-rabbit fluorescein-conjugated antibodies were from Zymed Laboratories Inc. (San Francisco, CA). Kainate, NMDA, and APV were from Sigma Chemical Co. Acknowledgments We are grateful to Dr. Thomas L. Leto for generously providing the a-fodrin cDNA probe and for discussion and to Dr. Dorothy E. Croall for the generous gift of anti-calpain I antisera. We thank Dr. Jon Morrow for his advice and support; Drs. Stuart CullCandy, Giulio Levi, Robert Balazs, and Dennis Choi for discussion; Drs. Pompeo Macioce and Claudio Giovannini for their collaboration; and Drs. Mark Mayer, Jon Morrow, and Andres Buonanno for critically reading the manuscript. We also thank Ms. Maxine Schaefer for typing the manuscript. This work was partially supported by a grant from the Italian National Research Council (grant 88.03251.04, Project “Signal Transduction Mechanisms in Nerve Cells’? to V. G. and by a NATO Research Grant (0195/88) to T. C. P. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

September

13, 1990; revised

January

11, 1991.

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