Neurochemical Research, Vol. 24, No. 2, 1999, pp. 249-254

The Phosphoinositide Signaling Cycle in Myelin RequiresCooperative Interaction with the Axon*

(Accepted August 21, 1998)

Previous studies on the origin of myelin phosphoinositides involved in signaling mechanismsindicated axon to myelin transfer of phosphatidylinositol followed by myelin-localized incorpo-ration of axon-derived phosphate groups into phosphatidylinositol 4-monophosphate and phospha-tidylinositol 4,5-bisphosphate. This is in agreement with other studies showing the presence ofphosphorylating activity in myelin that converts phosphatidylinositol into the mono-and diphosphoderivatives. It was also found that the second messenger, inositol 1,4,5-trisphosphate, is hydrolyzedto inositol 1,4-bisphosphate by a myelin-localized enzyme. The present study was undertaken todetermine the locus of the remaining reactions leading to formation of free inositol and completionof the cycle by resynthesis of phosphatidylinositol. The latter reaction was found to occur pref-erentially in isolated axons, and to a limited extent if at all in myelin. On the other hand, hydrolyticreactions which sequentially convert inositol 1,4,5-trisphosphate to inositol 1,4-bisphosphate, in-ositol 1-phosphate, and free inositol were found to occur more prominently in myelin. Thus,restoration of phosphoinositides following signal-induced breakdown of PIP2 in myelin is seen asrequiring metabolic interplay between myelin and axon.

KEY WORDS: Myelin; axon-myelin transfer; signal transduction in myelin; inositol phosphates; myelin phos-phoinositides; inositol phosphate hydrolases; phosphatidylinositol synthase; phosphoinositide signaling in my-elin.

Goutam Chakraborty,1 Anthony Drivas,1 and Robert Ledeen1,2

INTRODUCTION

Although there were early indications of myelin-localized enzymes in the form of cyclic nucleotide phos-phodiesterase (1), aminopeptidase (2), protein kinase(3,4) and phospholipase (5) activities, myelin was forsome time regarded as a largely inert membrane withlimited metabolic activity. The research of many labo-ratories up to the present time has changed that percep-tion, over 40 enzymes thus far having been identified asintrinsic myelin constituents. The studies of Kunihiko

1 Department of Neurosciences, New Jersey Medical School, UMDNJ185 South Orange Ave., Newark, NJ 07103.

2 To whom to address reprint requests Tel: 973-972-7989; Fax: 973-972-5059; E-mail: ledeenro@umdnj.edu

* Special issue dedicated to Dr. Kunihiko Suzuki

Suzuki and coworkers contributed importantly to thesedevelopments, beginning with the discovery of choles-terol ester hydrolase (6,7) and synthetase (6,8) activitiesin this membrane. They subsequently helped to the elu-cidate the properties of UDP-galactose:ceramide galac-tosyltransferase within the myelin sheath (9). The rather

Abbreviations: AEF, axon-enriched fraction; IP, inositol monophos-phate; IP2, inositol bisphosphate; IP3, inositol trisphosphate; PI, phos-phatidylinositol; PIP, phosphatidylinositol 4-monophosphate; PIP2,phosphatidylinositol 4,5-bisphosphate; IP-Pase, inositol 1-phosphatase;IP2-Pase, inositol 1,4-bisphosphate-4-phosphatase; IP3-Pase, inositoll,4,5-trisphosphate-5-phosphatase; DTT, dithiothreitol; AEBSF,4-(2-aminoethyl)benzene-sulfonyl fluoride; GFAP, glial fibrillaryacidic protein; NF-H. high molecular weight, 220 kDa component, ofneurofilament; HRP, horseradish peroxidase; TCA, trichloroaceticacid; PBS, phosphate buffered saline; TPBS, PBS containing 0.05%Tween-20.

2490364-3190/99/0200-0249$16.00/0 C 1999 Plenum Publishing Corporation

250 Chakraborty, Drivas, and Ledeen

surprising array of enzymes, receptors, and signaling ef-fectors known to exist in this membrane has been de-scribed in several reviews (10-14).

Several of the previously characterized myelin en-zymes are now known to be components of signal trans-ducing systems, associated receptors of which have beendetected in purified myelin (for review—ref. 14). Theseinclude cholinergic muscarinic receptors (15) which ac-tivate phosphoinositide phosphodiesterase with libera-tion of inositol 1,4,5-trisphosphate (IPS) anddiacylglycerol (16). The former second messenger wasshown to be actively hydrolyzed to inositol 1,4-bisphos-phate (IP2) within the myelin sheath (17), leaving un-resolved the locus of subsequent reactions required tocomplete the hydrolytic process with formation of freeinositol and resynthesis of phosphatidylinositol (PI). Thepresent study, undertaken to explore this question, hasrevealed that in addition to possessing IPS phosphatase(IPS-Pase) activity, purified myelin also has the capacityto hydrolyze both IP2 and inositol monophosphate (IP).However, it proved relatively ineffective in the synthesisof PI, a reaction that proceeded rapidly in isolated axons.These results, combined with previous evidence that my-elin phosphoinositide synthesis is dependent on trans-axonal migration of axonally transported substances(18), support the concept of coordinated interaction ofmyelin and axon in completion of the myelin-localizedphosphoinositide signaling cycle.

EXPERIMENTAL PROCEDURE

Materials. The following substances were purchased from thesources indicated: adult (30-40 day old) male Sprague-Dawley rats,from laconic Farms (Germantown, NY); myo-[2-3H(N)inositol (22.1Ci/mmol), D-inositol [2-3H(N) 1-phosphate (11.0 Ci/mmol), D-inositol[l-3H(N)]l,4-bisphosphate (10.0 Ci/mmol), D-inositol [1-3H(N)] 1,4,5-trisphosphate (21.0 Ci/mmol) from New England Nuclear (Boston,MA); unlabeled myoinositol, D-inositol 1-phosphate, D-inositol 1,4-bisphosphate, D-inositol 1,4,5-trisphosphate, CDP-diacylglycerol, am-monium formate, aprotinin, leupeptin, benzamidine, pepstatin, EDTA,dithiothreitol (DTT), 4-(2-aminoethyl)benzene-sulfonyl fluoride(AEBSF) and Kodak Biomax-MR2 film from Sigma Chemical Co. (St.Louis, MO); Accell-QMA Sep Pak cartridge columns from Waters,Millipore Corp. (Millford, MA); Ecoscint A and gel supplies fromNational Diagnostics (Atlanta, GA); monoclonal anti-glial fibrillaryacidic protein (GFAP) and polyclonal anti-NF-H (high molecularweight, 220 kDa component of neurofilament) antibodies from Chem-icon (Temecula, CA); goat anti-rabbit HRP and goat anti-mouse HRPfrom Cappel (Durham, NC); ECL™ detection kit and rainbow molec-ular weight protein standard mixture from Amersham (ArlingtonHeights, IL); most other chemicals from CMS Inc. (Houston, TX).

Preparation of Brain Fractions. A 10% homogenate of wholebrain (4/group) was prepared in 0.3 M sucrose containing 20 mM Tris-HC1 (pH 8.0), DTT (2 mM), and the following mixture of proteaseinhibitors: leupeptin (10 |0.g/ml), aprotinin (1 ug/ml), AEBSF (O.2

mM), EDTA (1 mM), benzamidine (1 mM), and pepstatin (10 Ug/ml)(solution A). Microsomes were prepared as previously described (19),beginning with homogenization in solution A and centrifugation at 18kg for 10 min. The supernatant was centrifuged at 105k g for 60 minto separate the microsomal pellet from the final supernatant. The for-mer was divided into portions and each washed 2x in the buffer cor-responding to the enzyme assay to be performed (see below). Toobtain myelin, the above 18k g pellet was prepared as a 5-10% hom-ogenate in solution A and processed according to Norton and Poduslo(20) as modified by our laboratory (21) for greater purity.

An axon-enriched fraction (AEF) was isolated from rat brainstems by the method of DeVries et al. (22) as later modified (23). Allsucrose solutions used during preparation of AEF were buffered with50 mM K-phosphate, pH 7.5, containing 150 mM NaCI. Brain stemtissue was finely minced and suspended in 100 volumes of 1.2 Msucrose containing the above buffer. This was dispersed with 6 strokesof the loose pestle followed by 6 strokes with the tight pestle in aDounce homogenizer (Kontes, NJ). The homogenate was centrifugedat 82,500 g for 15 min and the floating layer resuspended by mildhomogenization in 1.0 M sucrose followed by centrifugation at 82,000g for 15 min. The floating layer was resuspended by mild homoge-nization in 0.85 M sucrose with above buffer (solution B) and centri-fuged as above. The final floating layer was suspended in 50 mMK-phosphate buffer pH 7.5 (1 ml/30 ml of original homogenate) with3-4 strokes of the loose pestle, and kept at 4°C with constant stirringfor 2 hrs. The suspension was made approximately 0.85 M sucroseand 0.15 M NaCI by adding solid sucrose and NaCI, and the mixturecentrifuged at 82,500 g for 10 min. The axonal pellet was rehomo-genized in solution B and centrifuged at 82,500 g for 10 min, and thelatter procedure repeated 4X. After the second or third centrifugationthere was usually no floating myelin observed. All procedures werecarried out at 4°C and the above-mentioned mixture of protease inhib-itors was employed throughout.

Enzyme Assays. PI synthase (EC 2.7.8.11) was assayed essentiallyas described by Antonsson (24). The reaction mixture, in 0.2 ml, con-tained 50-100 jug protein; reaction was terminated by addition of 2.0ml of methanol-water (2:1, v/v) and [3H]PI was isolated by anion ex-change chromatography with Accell-QMA Sep-Pak cartridges (see be-low). Myoinositol-1-phosphatase (IP-Pase) (EC 3.1.3.25) wasdetermined according to Gee et al. (25) with minor modifications. Theassay mixture contained 50 mM Tris-HCl pH 8.0, 3 mM MgCl,, 0.3%Triton X-100, 1.0 mM [3H]IP (27 pCi/mmol) plus 250-350 ug protein.Reaction mixtures were incubated with shaking at 37°C for 15 min.The reaction was terminated by addition of 2.0 ml methanol-water (2:1, v/v) and the resulting [3H]myoinositol separated by anion exchangechromatography (see below). Inositol-( 1,4)-bisphosphate-4-phospha-tase (IP2 -Pase) was determined according to Ragan et al. (26). Thereaction mixture, containing 250-350 \ig protein, was essentially thesame as that for IP-Pase (see above). Reaction was stopped by additionof TCA, followed by removal of the latter with ether extraction andpH adjustment to 7.0 with 1 N NaOH and 0.3 mM borax(Na2B4O7-10H,O). In some runs 10 mM LiCl was added to inhibit IP-Pase, but better results were obtained without this addition; in thatcase reactivity was based on the sum of [3H]inositol and [3H]IP whichwere isolated by anion exchange chromatography (see below). Inositol1,4,5-trisphosphate 5-phosphatase (IP3-Pase) (EC 3.1.3.56) was assayedas previously described (17), with inclusion of 10 mM LiCl. The reac-tion was stopped by addition of TCA, the latter removed by ether ex-traction and the pH adjusted to 7.0 as above. The reaction products wereseparated by anion exchange chromatography (see below) and reactivitycalculated on the basis of [3H]1P and [3H]IP2. In this and the above 2assays, some reaction mixtures contained 125 mM KC1.

Phosphoinositide Signaling Cycle in Myelin 251

Fig. 1. Purity of myelin and axon-enriched fraction. Following SDS-PAGE, proteins were transferred electrophoretically to PVDF mem-brane. The latter were probed with (A) rabbit polyclonal anti-NF-Hand (B) mouse monoclonal anti-GFAP antibody. After thorough wash-ing the membranes were treated with corresponding HRP-conjugatedsecond antibodies, followed by ECL detection. Lane identification: 1,purified myelin; 2, axon-enriched fraction; 3, microsomes, Each lanehad approximately 25 jig protein. Left margins indicate molecularweight standards (kDa).

Ion-Exchange Separation of Products. This chromatography em-ployed Accell QMA Sep-Pak cartridges, following the procedure ofWreggett and Irvine (27) as applied to this enzyme (17). This em-ployed gradients composed of increasing concentrations of the ternarymixture, ammonium formate-formic acid-borax. The principal modi-fication was that methanol-water (2:1, v/v) was employed as solventfor the above ternary mixtures in assay of Pl-synthase and IP-Pase,whereas water alone was used as solvent for the ternary mixtures inassay of IP2-Pase and IP3-Pase as originally described. Fractions of 4ml, 4 ml, 2 ml, and 2 ml were collected in each case; similar stepwiseelutions were performed with 12 ml methanol-water (2:1, v/v) for elu-tion of PI and IP. 1 ml aliquots of each fraction were mixed with 6ml Ecoscint A for counting with a Tricarb 2100 TR Packard liquidscintillation spectrometer.

Immunoblot Analysis. Protein separation was carried out withSDS-PAGE according to Laemmli (28), using 8% acrylamide gels, 1mm thick, in a mini-protein gel apparatus. After separation the proteinswere transferred electrophoretically to a polyvinylidene difluoridemembrane. Nonspecific binding to the membrane was blocked by in-cubating with 4% dry nonfat milk in PBS containing 0.05% Tween-20 (TPBS) overnight at 4°C. The membrane was then exposed to eitheranti-GFAP (1:750 dilution) or anti-NF-H (1:1000 dilution) antibody inblocking solution for 3 hrs at room temp with constant shaking. Afterwashing with TPBS, the membrane was incubated with the corre-sponding second antibody tagged with horseradish peroxidase (HRP)(1:1000 dilution in blocking solution) for 1 hr at room temp. Themembrane was washed extensively with TPBS. Immunoreactive bandswere visualized with Biomax MR-2 film, using ECL™ detection kitaccording to manufacturer's protocol. Protein estimation was carriedout according to Lees and Paxman (29).

RESULTS

Myelin purity is a critical factor in studies designedto reveal the presence (or absence) of myelin-localizedenzymes. Although the purity of myelin isolated by theemployed procedure was previously demonstrated(19,21), additional evidence was adduced in the presentstudy in the form of Western blot analysis (Fig. 1). This

indicated virtually complete absence of neurofilamentprotein NF-H and GFAP, markers for neuron and astro-cyte cytoskeleton proteins, respectively. The axon-en-riched fraction, on the other hand, showed an abundanceof NF-H and a modest amount of GFAP. The latter hasbeen attributed to the strong tendency of GFAP to ad-sorb adventitiously to membranes (29a).

The 3 enzymes involved in hydrolysis of IP3 to freeinositol were all present in purified myelin at measurablelevels which were higher than the corresponding activ-ities observed in AEF (Table I). The IP-Pase activity inmyelin was approximately 67% of that in microsomesand 5-6X higher than that in AEF, indicating myelin asa more likely locus of this reaction than the axon. IP3-Pase activity was also relatively high in myelin, andcomparable to the previously reported value for this en-zyme (17), although in this case the AEF also has con-siderable activity. IP2-Pase activity was alsosignificantly higher in myelin than in AEF when the re-action was carried out in the absence of LiCl (Table I).The latter salt has been used for assay of IP3-Pase andIP2-Pase because of its ability to inhibit IP-Pase (30),but its reported ability to also inhibit IP2-Pase (26) madeit unsuitable for use in the assay of this enzyme in thepresent study. We have found that both the myelin- andmicrosome-associated forms of this enzyme are stronglyinhibited by LiCl, these values being 3.15 ± 0.39 and42.3 ± 8.81 nmol/mg protein/hr, respectively (compareto Table I). On the other hand IP2-Pase in AEF wasrelatively unaffected by LiCl. We also observed that IP-Pase and IP2-Pase activities occurred predominantly inthe soluble fraction (not shown), in accord with a pre-vious report (26). The low levels of IP-Pase and IP2-Pase in AEF, compared to those in microsomes, suggeststhose measured values may reflect extra-axonal contam-inants. In most cases enzyme activity was significantlyelevated by addition of KCl; this was believed to reflecta general salt effect since approximately the same effectwas observed when IP3-Pase was measured in the pres-ence of an equimolar concentration of NaCl (not shown).Use of assays with and without salt served to highlightdifferences in some cases between myelin enzymes andthose of other fractions (e.g., the modest KCl-inducedincrease in myelin IP-Pase vs the 4.7-fold increase ofthat enzyme in axons).

In contrast to the inositol phosphate hydrolytic en-zymes which showed higher activity in myelin thanAEF, PI synthase was found to be roughly 6X as activein AEF as in myelin (Table II). The fact that myelinshowed approximately 5% the activity of microsomesmay indicate the level of contamination of the former,although this might be considered an upper limit in view

252 Chakraborty, Drivas, and Ledeen

of the low levels of contaminants that were previouslyobserved in myelin prepared by the present procedure(19,21).

DISCUSSION

Phosphoinositides are relatively abundant in myelincompared with other neural membranes (31-33) andtheir phosphomonoester groups were shown to undergoactive turnover in this membrane (34-36). Myelin phos-phoinositides rapidly incorporate 32PO4 after injection ofthe latter into rat brain or application to brain slices,accounting for a high percentage of radioactivity amongtotal brain or myelin phospholipids (37,38). Myelinphosphoinositides were also shown to become labeledthrough axon-to-myelin transfer of substances undergo-ing axonal transport in the rabbit optic system (18). Inthis process 2 mechanisms of transfer were discerned:(a) transfer of intact lipid, in the case of PI, and (b)transfer of phosphate groups which became enzymati-cally linked to the 4- and 5-positions of inositol in my-elin PIP and PIP2. Thus, the latter 2 lipids in myelinfrom optic tract and superior colliculus became increas-ingly labeled between 7 and 21 days following intraoc-ular injection of 32PO4. Enzyme activities which catalyzesuch phosphomonoester formation were previously de-tected in isolated myelin (39).

Axon-myelin transfer of PI was indicated in anelectron microscopic study of Gould (40) in which ax-onal PI, labeled by injection of [3H]inositol into sciaticnerve, caused progressive increase of radioautographicgrains over myelin coincident with decline of axon-lo-calized grains. Additional indications of intraxonal syn-thesis of PI have been reported (41,42) includingevidence for axonal transport of CDP-diacylglycerol: in-ositol transferase (PI synthase) (43). These results arecongruent with the present biochemical evidence for ap-preciable PI synthase activity in AEF and, in conjunctionwith the relatively low level of PI synthase activity ob-served here in purified myelin, suggest the axon as onelikely source of myelin-localized PI. Myelin phosphoi-nositides might also derive in part from the oligoden-drocyte, considering the evidence for continuous flow oflipids from glial cell bodies into the lamellae, even formature myelin (44,45). This raises the possibility of dif-ferent pools of phosphoinositides within myelin, withcorresponding specificity of supply routes.

A second messenger-generating role for myelinphosphoinositides was indicated in the observations thata muscarinic agonist and a nonmetabolizable analog ofGTP stimulated formation of IP3 through breakdown ofPIP2 in myelin (16,46,47). Muscarinic receptors whichmediate this reaction as well as the inhibition of aden-ylate cyclase were shown to occur in highly purified my-elin (15). In addition a variety of GTP-binding proteinshave been detected (48,49) along with PIP2 phospho-diesterase (16,50,51) and several other enzymes in-volved in the metabolism and synthesis ofphosphoinositides (for review, ref. 14). The presence of3 inositol phosphate-metabolizing enzymes in myelin,observed in the present study, shows that myelin-local-ized enzymes have the capacity to terminate the IP3 sig-nal and begin the recycling process by stepwisehydrolysis of IP3 to free inositol. The results of thisstudy have also indicated that myelin lacks the capacityto complete the cycle by incorporating the liberated in-ositol into PI, a reaction that can be carried out in the

Table I. Distribution of IP-Pase, IP2-Pase and IP3-Pase Activity" in Subcellular Fractions of Rat Brain

MyelinAxonMicrosoraes

IP-Pase

-KCl

92.8 ±6.6316.3 ±0.8138±14.0

+KCl

138±14.077.0 ±24.0259 ±24.0

IP2-Pase

-KCl

40.0 ±2.01c

18.5 ±1.52b

172 ±10.9

+KCl

52.6±2.89c

29.6 ±2.89b

166 ±12.4a

IP3-Pase

-KCl

481 ±48.3b

334 + 40.02141 ±169b

+ KCl

928 ±132b

701 ±90.02880 ±346b

Axon enriched fractions were isolated from pooled brain stem whereas all other fractions were generated fromtotal brain.a Specific activity is expressed as nmol/mg protein/hr. Values are expressed as mean ± SEM (n = 3for all the values except for the followings: b, n = 4; c, n = 5 and d, n = 2, i.e., range rather than SEM)

Table II. Comparison of Phosphatidylinositol Synthase Activity inSubcellular Fractions of Rat Brain

Fraction

MyelinAxonMicrosomes

Specific Activitya

32.5 ± 1.34 (n = 3)203 ± 11.2 (n = 3)614 ± 3.23 (n = 5)

Axon enriched fraction was prepared from pooled brain stem and otherfractions were prepared from whole brain. Each fraction was allowedto stand overnight at 0 °C in the presence of protease inhibitor cocktailprior to enzyme assay. " Specific activity is expressed as nmol/mgprotein/hr. Values are expressed as mean ± SEM.

Phosphoinositide Signaling Cycle in Myelin 253

Fig. 2. Proposed mechanism of phosphoinositide cycle involving concerted action of axon and adjacent myelin sheath (revised from Fig. 2 of ref. 18). PIsynthase, previously shown to enter the axon through axonal transport (43), catalyzes synthesis of PI that enters myelin and is converted there to PIP andPIP2; the incorporated phosphate is postulated to derive from the axon, following phosphatase hydrolysis of axonally transported phosphoproteins and/orphospholipids. Upon activation of the cholinergic receptor, shown to be present in myelin (15), PIP2 is broken down to the second messengers DAG andIP3. The latter is sequentially hydrolyzed within the myelin membrane to free inositol, which enters the axon for recycling into PL The latter has beenshown to undergo axon-to-myelin transfer (40), possibly aided by phospholipid exchange protein(s) that reacts with myelin (53-55).

axon. The PI thus formed could conceivably enter my-elin via phospholipid exchange proteins which havebeen demonstrated to be capable of transferring PI tomyelin from other membranes (53-55).

The model suggested by these findings is thus onein which full operation of the phosphoinositide signalingreactions in myelin requires coordinated interaction withthe axon (Fig. 2). This proposed mechanism, with IP3hydrolysis to free inositol occurring entirely within my-elin, differs somewhat from that proposed earlier (18),according to which only the first step—hydrolysis of IPSto IP2—ocurred in myelin. The latter assumption wasbased on the observation (17) that IP2 was not hydro-lyzed to IP in myelin under the conditions of that ex-periment; however, the present study has shown thatresult to have been artifactual due to the presence ofLiCl which inhibits IP2-Pase as well as IP-Pase (26).Omission of LiCl revealed a relatively high level of IP2-Pase in myelin compared to the axon (Table I). The pres-ence of a signaling mechanism involving both myelinand axon can be viewed in the context of other indica-tions that axons and oligodendrocytes (including myelin)communicate in an ionically-mediated fashion (56), andthat material transfer occurs between these cellular com-partments (57-59).

ACKNOWLEDGMENTS

This work was supported by NIH grant NS16181. The technicalassistance of Dr. Ratna Reddy is gratefully acknowledged.

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