- Email: [email protected]
The expression of an N-CAM serum fragment is positively correlated with severity of negative features in type II schizophrenia
BIOL PSYCHIATRY 1988:23:169-775
769
The Expression of an N-CAM Serum Fragment is Positively Correlated with Severity of Negative Features in Type II Schizophrenia Finbar Lyons, Mary L. Martin, Carol Maguire, Anne Jackson, Ciaran M. Regan, and R. K. Shelley
The expression cfa 70-kD serurtt,frugment gf the neural cell adhesion molecule (N-CAM) in schizophrenia is described. Schixphrenic putients (n = 40) werefound to huve stutistically significant increases Ip < 0.0001) in serum N-CAM levels when compared to normal individuals (n = 26), und this could not be ussociated wirh age or .sex. This dtjferencr was more murked (p < 0.0001) between type II xhizophrenics (n = 13) and normal individuals (n = 26) than when paGnt.s in the overlap group between type I and @pe I! .s~hizo~hrenia (n = 18) urere compared to normal individu~l,s f~ < O.OOl]. This d~~er~n~e rerna~n~d significant (F < 0.01) peahenoverlap parients were ~~rnp~tr~d to those of type If .s~~li~a~~lrenia. ~ur~~lertn~~re, ~s~h~~~jp~lrgtl~~ patients with lower sc’rum N-ChM proved lo be belter, r~sp[~nder~s fo neurolepri~ therapy. We suggesr that rhesr ~Jl~~~i~te~~ xtwm N-CAM fcvefs reflect an kreased synaptic turnover rate in this pyvchotic sfafe.
Introduction Despite much research, the causes underlying schizophrenia remain obscure. The recent classification of schizophrenia into two syndromes (Crow 1980), type I and type II, has been a productive source of new hypotheses on the etiology of this disorder (Shelley and Walsh 1985). Type I schizophrenia is characterized by positive symptoms (delusions, hallucinations, and thought disorder), which may correlate positively with an increase in brain dopamine receptors (Owen et al. 1985). Type II schizophrenia is associated with negative features (loss or absence of affect, poverty of speech, and loss of volition), which are more likely to be associated with intellectual impairment and structural changes in the brain, as shown by computed tomography and postmortem investigation (Crow et al. 1981). These two syndromes are not exclusive, and overlap may occur in 40% of patients with schizophrenia. The possible role of altered neuronal plasticity, mediated by the formation and degeneration of synapses (Cotman and Nieto-Sampedro 1982, 1984) in the etiology of schizophrenia has only recently been considered (Haracz 1985). An example of this is
770
BIOL PSYCHIATRb 1988:23:769-?I5
postlesional plasticity, where compensatory reinnervation of uninjured afferenth or cprouting results in functional synapses (Nieto-Sampedro et al. 1982). There are over 30 studies of the use of computed tomography in schizophrenia. The finding of enlarged ventricles in most of these suggests a subgroup with neuronal cell loss (Johnstone 1985). This group most resembles Crow’s type II. Of further interest is the finding of increased density of catecholaminergic nerve terminals in some brain areas of patients who had schiz,ophrenia, and this hyperinnervation has been suggested to result from a primary lesion nearby (Hokfeldt et al. 1974; Coyle and Johnston 1980). Here, we have explored the pobstbility of increased synapse turnover being associated with schizophrenia by using 11 \e-rum fragment of the neural cell adhesion molecule (N-CAM). N-CAM is a developmentally regulated cell surface sialoglycoprotein that IS believed to be integrally involved in the structuring and final elaboration of the central nervous system (Jorgensen 198 I; Meier et al. 1984; Choung and Edelman 1984; Edelman 1986). The protein is a complex of three immunologically related polypeptides of 200. 140, and 115 kD molecular weight (Choung and Edelman 1984; Sheehan et al. 1986). Cell adhesion is believed to be mediated by a homophilic binding between N-CAMS on apposing cells (Brackenbury et al. 1977). The strength of this adhesion is dependent on the amount of sialic acid on each molecule (Hoffman and Edelman 1983), and as this decreases during development (Jorgensen 198 1; Rothbard et al. 1982; Choung and Edelman 1984: Meier et al. 1984), increased adhesivity occurs and eventual histogenesis. Recent studies have suggested that in addition to its developmental function. N-CAM may also play a role in the regeneration and restructuring of the mature neuromuscular junction (Covault and Sanes 1985. 1986; Rieger et al. 1985: Daniloff et al. 1986). It is also reasonable to assume that such events occur centrally, as here the neuronal circuitry is being constantly remodeled (Cotman and Nieto-Sampedro 1982, 1984; Nieto-Sampedro et al, 1982). Given that N-CAM has been demonstrated to be sloughed from the cell surface (Thiery et al. 1977; Annunziata et al. 1983), and that this soluble form has also been shown to be present in cerebrospinal fluid and serum (Jorgensen and Bock 1975: Ibsen et al. 1983), several workers have attempted to correlate changes in ~&ublc NCAM expression with altered states of synaptic function in psychotic and neurodegenerative conditions of the central nervous system (Jorgensen and Bock 1975; Jorgensen et al. 1977, 1980, 1982; Soelberg Sorensen et al. 1983). Although no significant correlations were found in these studies, this can be related to the low levels (<4 pgiml) of N-CAM found in human serum and cerebrospinal fluid (Ibsen et al. 1983) and which cannot be readily detected using crossed immunoelectrophoresis (Jorgensen and Bock 1975). Thus, we have considered it worthwhile to reappraise this situation using a sensitive cnzymelinked immunosorbent assay to compare soluble N-CAM levels in the serum (~1schizo-phrenic and control populations.
Methods Patients (26 men, 14 women) were drawn consecutively from a general hospitai clinic (St. James’ Hospital, Dublin) of a community-orientated psychiatric service that they attended for regular depot neuroleptic medication. Forty patients met the inclusion standards of fulfilling Feighner’s criteria (Feighner et al. 1972) for schizophrenia, were aged 18-65 years, gave valid consent, and had no concurrent physical illness. Twenty-six controls (7 men, 19 women) were selected from physically and psychologically normal hospital staff. The mean patient age was 36 years ( f 10.1) and that of the control group
N-CAM Serum Fragment
and Negative Symptoms
BIOL PSYCHIATRY 1988;23:769-775
111
was 33 years ( + i 1.2). The difference between these means is not statistically significant (Student’s 1 = 1.34, df = 64). The patient group was classified into type I (positive symptoms only), type II (negative symptoms only), or overlap type I/Ii (both positive and negative symptoms). The definition of positive and negative subtypes followed was that of Andreasen and Olsen (1982). Only 4 patients could be classified as type I; agreement could not be reached on 5, and in the case of 2 of these, this was because of associated alcohol abuse and mental handicap. Finally, a clinical estimate was made of whether or not patients could be deemed to have responded well to neuroleptic medication. Fourteen were thought to have a good therapeutic outcome, 21 a poor outcome, leaving 5 on whom agreement as to outcome could not be reached. Blood samples were obtained by v~nipuncture and were allowed to clot before the serum was prepared by centrifugation at 3000 rpm for 10 min. N-CAM levels were estimated blindly, using an enzyme-linked immunosorbent assay. Briefly, serum samples were diluted 1:40 in Dulbecco’s phosphate-buffered saline (DPBS), and 50-p] aliquots were allowed to absorb to flat-bottom wells of microtiter plates (A/S Nunc, Roskilde, Denmark) for 18 hr at 4°C. These were then washed 3 times with DPBS containing 0.3% Tween-20, (Sigma Chemical Co., St. Louis, MO), and 50~1 aliquots of an N-CAMspecific antibody (Sheehan et al. 1986), diluted I:20 with DPBS containing 0.05%’ Tween20, were added to each well and incubated for 90 min at room temperature. The wells were again washed and then incubated with 50-pl aliquots of a peroxidase-conjugated sheep anti-rabbit IgG,A, and M (Serotec Ltd.), diluted 1:250 with DPBS containing 0.05% Tween-20. The wells were again washed, and SO-*1 aliquots of Sorensen’s buffer (50 rnM Na2HP04, 25 mM citric acid, pH 5.0), containing the peroxidase substrate ophenylenediamine (0.4 mgiml; Sigma) and HzOz (0.5 pgiml; Sigma) were added to each well. The reaction was allowed to proceed for 5 min before the color was enhanced by the addition of 100 ~1 of 1N H,SO,. The color was read at 492 nm, and the results were expressed in arbitrary units (AU), 1 AU being equivalent to an optical density unit/ microgram protein. Protein concentrations were estimated according to the procedure of Lowry et al. (195 l), and no difference in serum protein content was noted between normal and psychotic individuals. Statistical differences were estimated using an unpaired Student’s r-test. The molecuiar weight of the serum N-CAM fragment was determined using previously described immunoblo~ing procedures with an N-CAM-specific polyclonal antibody (She&n et al. 1986).
Resultts Immunoblotting procedures demonstrated serum N-CAM to comprise a 70-kD fragment, and no evidence of other immunoreactive fragments was obvious (Figure 1). Schizophrenic patients were found to have highly significant increases (p < 0.0001) in serum N-CAM levels when compared to normal individuals (Table l), and this could not be related to differences in age or sex. This difference was more marked between type II schizophrenics and normal individuals (p < O.OOOi) than that of patients in the overlap type I/II group and normal individuals fp < 0.001) (Table 1). Thus, patients with only negative features have higher N-CAM serum levels than patients with mixed positive and negative symptoms. This difference also remained significant (p < 0.01) when overlap patients were compared with those of type II schizophrenia (Table 1). When patients were classified
112
BIOL PSYCHIATK) 1988;23:769-775
Figure I. lmmunoblot demonstrating the molecular weight of the N-CAM serum fragment. The sample, containing SO p,g protein, was separated on a 10% acrylamide gel.
Table I. Serum N-CAM Levels in Schizophrenics and Normal individuals Schizophrenics
N0lTIKTl All
Population size Serum N-CAM units Significance
26
40
x.7 rt 0.22
10.01 to. 16
p ( ~.~~
Values are expresseda%arbitrary unit\ and nn! the mean % XSI.
Type l/II
---__I Type
11
Table 2. Relationship
173
BIOL PSYCHIATRY 1988:23:769-775
N-CAM Serum Fragment and Negative Symptoms
between Serum N-CAM Levels and Therapeutic
Response Response Poor
GOOd
?I
14
Population size Serum N-CAM
units
IO.4
9.1 2 0.18
+ 0.23
Values are expressedas mean * SEMand are significantlydifferent (0.02 > p > 0.01).
according to clinical response to neuroleptic medication, those with a poor therapeutic outcome had significantly higher serum levels (p < 0.02) (Table 2).
Discussion The serum fragment of N-CAM was demonstrated to be 70 kD in molecular weight, and this is very similar to the 6.5kD fragment reported to be generated by spontaneous proteolysis of purified N-CAM (Hoffman et al. 1982). Furthermore, this fragment contains all the antigenic determinants that neutralize the cell adhesion blocking activity of antibodies prepared to intact N-CAM (Hoffman et al. 1982). Consequently, this serum fragment may represent the functional amino-terminal binding domain of the molecule, which would be involved in synapse formation, and an increased release of this could therefore be associated with increased synapse turnover. A possible explanation for elevated levels of N-CAM in the sera of schizophrenics is that neuroleptic therapy may damage neuronal and/or muscle membranes or directly interfere with the enzyme-linked immunosorbent assay (ELBA) solid-phase immunoassay. However, no change in serum N-CAM levels was noted in rats 2 weeks after a depot injection of flupenthixol decanoate or fluphenazine decanoate at twice the concentration given to the patients, and a tenfold excess of therapeutic serum neuroleptic levels (Seeman 1977) was found to have no effect on the ELBA immunoassay procedure (data not shown). Furthermore, in a small but representative group of motorneuron disease patients (n = S), no increase in serum N-CAM was evident (data not shown), suggesting that elevated levels of this protein do not reflect neurodegenerative changes. These results indicate that N-CAM levels can distinguish between a group of normal individuals and patients with a diagnosis of schizophrenia and also between subtypes of this psychotic disorder. However, they must be interpreted with caution. Although there is evidence that N-CAM is involved in synaptic st~cturing and reorganization (Covauft and Sanes 198.5, 1986; Rieger et al. 1983, it is not clear that this would result in an increased release of soluble N-CAM, as would be reflected in serum levels. if this proves to be the case, then natural synaptic turnover rate (Cotman and Nieto-Sampedro 1982, 1984) must be increased and sustained in schizophrenia, and markedly so in type 11 schizophrenia. We gratefully
appreciate the assistance of Mr. Roddy Monks in the preparation of this manuscript.
References Andreasen 794.
NC, Olsen S (1982): Negative v. positive schizophrenia.
Arch Gen ~~~~~~zjff~~35:789-
Annuntiata P, Regan CM. Balazs R f 1983): Development of cerebetlar celis in neuron-enriched cultures: Cell surface proteins. Dev Brain Res 8:2&i-273.
774
6lOL PSYCHIATRY 1988;23:769-77s
Brackenbury R. Thiery J-P. Rutishauser U. Edelman GM (1977): Adhesion among neu;$ CC& ol the chick embryo. I. An immunologi~af assay for molecules involved in ccli-ceit tvnding. .: Rid Ckm 252:68X-6840. Chuong C-C. Edelman GM (1984): Alterations in neural ccl1 adhesion molecuics dtic,itg deuct opment of different regions of the nervous qystcm. J h’eumsci 4:2X4--2368. M C19821: Brain function. synapse renewal and plastictr!
Cotman CW, Nieto-Sampcdro Psyhol 3337 I-401
Cotman CW. Nicto-Sampedro
.‘~wI~KPI,
M I IYX4): Cell biology ot synaptic plasticrty. .Sc,rr,rc,!j.:I’< 17x7.
120-l Coyle JT. Johnston MV ( 1980): Functional hyperinnervation of cerebral cortex by noradrcaergic neurons results from fetal lesions: Parallels with schizophrenia. Psyhophtharmnd 8rtll 16:1779.
Covault J. Sanzs JR ( 1985): Neural celi adhesion ~ll~)le~ule (N-CAM) accumulates and paralyzed skeletal muscles. Proc~ NLII~At& Sci USA 82:4544454X. Covauh J. Sanes JR (1986): Distribution of N-CAM in synaptic and extrasynaptic developing and adult skeletal muscle, .I Cell Bio/ 102:716-730. Crow TJ ( 1980): Molecular J 280:66-6X. Crow TJ. Corsellis
pathology
of schizophrenia:
JAN, Crash AJ. Frith CD, Johnstone
rn ~~f~er~~~t~d portions or
More than one disease concept:
Nt- Mt4
EC. Owen F. Bloxharn C, f.crrlcc- IN.
Owens DGC ( 198 I 1: The search for changes underlying the type II syndrome in schizophrenia. In Penis C. Struwe G. Jansson B (cds). Bio/qiccrl Psyhintp. Amsterdam: Elsevier!North.
Holland Biomedical
Press, pp 727-.73 I
Danilof’f JK. Levi G, Grumet M, Kieger F, Edelman GM (1986): Altered el;presblc,ri ut neurona: cdl adhesion molecules induced by nerve injury and repair. .I Crib Bkrl 103:929-945 Edelman GM ( 1986): Cell adhesion molecules in the regulation of animal form and tisstrc patrcrn. A~7~~~r Ret. Cull Bid 2:81-l 16. Feighner JP, Robins E, Guze SB, Woodruff RF, Winokur G, Munor R f 1973): &agnostic for use in psychiatric research, Awh Get? Psychirzt~ 76:X---63. Haracz JL ( 1985): Neuraf plasticity
in schizophrenia.
trlteria
Schir-ophr B&i I I : 101-X!(3.
Hoffman S, Sorkin BC, Brackenbury R, Mailhammer R, Rutishauser U, Cunningham Bc’. Edelman GM (1982): Chemical characterisation of a neural cell adhesion molecule purified from embryonic membranes. J Bid Chem 257:7720-7729. Hoffman S, Edelman GM (1983): Kinetics of homophilic binding by embryonic and adult iorms of the neural cell adhesion molecule. Proc NatI Ad Sci USA 80:5762--5766. Hokfeldt T, Ljundahl A, Fuxe K. Johansson 0 (1974): Dopamine nerve terminals in the rat hmbic cortex: Aspects of the dopamine hypothesis of schizophrenia. Science 184:177-- 1%. Ibsen S. Berczin V, Norga~d-Pedersen 13. Bock E ( 19831:enzyme-linke~i of the D2-gly~oprotein . J ~~~~~~~~~2~~~ 4 I :356-X2. Johnstone
EC (1985): Structural changes
cho~pharmarnlogy:
Xecrnr
Adwnws
in the brain in schizophrenia. Prospecfs.Oxford:
and Futurr
iI~rnun~~s~~rb~~~~ assay
In lversen SD ted,, PI.!Oxford Medicai Publica-
tions, pp 196-203. Jorgensen OS ( 198 I ): Neuronal 37:939-946.
membrane
DZ-protein during rat brain ontogeny.
.I .~‘~uro&rrr
Jorgensen OS, Bock E (I 975): Synaptic plasma membrane antigen 02 measured in hUmin cercbrospinal fluid by rocket immunoelectrophoresis. Determination in psychiatric and neurological patients. &and .I immunol 425-30. Jorgensen
OS.
cerebrospinal
Bock
E.
Beth
P.
Rafi~elsen
Huid of manic-melancholic
Jorgensen OS, Hemmingsen
synaptic turnover 61:3.56-X4.
OJ (1977): Synaptic membrane protern D2 In the patients. Aeia Ps~chiutr &WZ~ 56:%-56.
R, Kramp P, Rafaefsen OJ (1980): Bled-bran barrier selectivity and during delirium tremens and related clinical states. Artu P.s!‘r*hinrr &XZ~
N-CAM
Serum Fragment
and Ncgativc
775
BIOL PSYCHlrZTRY 1988;1.1m-775
Symptoms
Jorgensen OS, Reynolds GP. Riederer P, Jellinger K ( 1982):Parkinson’s disease putamen: Normal concentration of synaptic membrane marker antigens. J ~eu~~il ~ru~.~~j.~~~54: 17 l-l 79. Lowry OH, Rosebrough NJ. Farr AL, reagent. J Bid Chem 193:X15-275. Meier E. Regan CM. during development
Randall
RJ (1951):
Protein
measurement
with
the Folin
Balazs R (1984): Change in the expression of a neuronal surface protein of cerebellar neuroncs in vivo and in culture. J Neuroc&~m 43: 132X-I 335.
Nicto-Sampedro M. Hoff SF. Cotman CW ( 1982): Perforated synaptic densities: mcdiatcs in synapse turnover. Proc Nnrl Autd Sci USA 7957 18-5722.
Probable
intcr-
Owen F. Crawley J. Cross AJ. Crow TJ. Oldland SR. Poulter M. Veal1 N, Zanelli GD (I985): Dopamine D2 receptors and schizophrenia. In lversen SD (ed). P.~~c,hophnrrnrlc,cti~~~~~: Recw~r Ad~wrzc~~strrzd F~tt~rc Prospec~t.c. Oxford: Oxford Medical Publications. pp 2 16-227 Ricgcr F. Grumet M, Edeiman Crli Biof 101:285-293.
GM (1985):
N-CAM
at the vertebrate
neuromuscular
junction.
.I
Rothbard JB. Brackenbury R. Cunningham BA. Edelman GM t 1982): Differences in the carbohydrate structures of neural ccl1 adhesion molecules from adult and embryonic chicken brains. J Hrc~i C/tern 257: I I .064-I I .Ohl). Sccman P ( 1977): Anti-schizophrenic 26:1741-1748.
drugs. Membrane receptor sites
ofaction. Bid7m7
Plrtrrmc7u~l
Sheehan MC. Halpin CI. Rcgan CM. Moran NM, Kilty CG (1986): Purification and characterization of the D2 cell adhesion protein. Analysis of the postnatally regulated polymorphic forms and their cellular distribution. Ne~~ochrm Res I I : 1333-l 346. Shelley RK. Walsh N (1985): The btochemistry 133.
of schizophrenia-A
review.
Irish Mrd J 7X: 139-
Soelberp Sorensen P. Gjerris F, lbscn S. Bock E (19X3): Low cercbrospinal fluid c~)ncentrati~)n of brain-specific protein D2 in patients with normal pressure hydrocephalus. J ~‘~~~~~~~ SKY6X965. Thicry J-P, Brackenbury R. Rutishauscr U. Edelman GM (1977): Adhesion among ncurai cells of the chick embryo. Ii. Puritication and characterization of a cell adhesion molecule from neural retina. J Biol Chrrn 252:6X31--6X35.
769
The Expression of an N-CAM Serum Fragment is Positively Correlated with Severity of Negative Features in Type II Schizophrenia Finbar Lyons, Mary L. Martin, Carol Maguire, Anne Jackson, Ciaran M. Regan, and R. K. Shelley
The expression cfa 70-kD serurtt,frugment gf the neural cell adhesion molecule (N-CAM) in schizophrenia is described. Schixphrenic putients (n = 40) werefound to huve stutistically significant increases Ip < 0.0001) in serum N-CAM levels when compared to normal individuals (n = 26), und this could not be ussociated wirh age or .sex. This dtjferencr was more murked (p < 0.0001) between type II xhizophrenics (n = 13) and normal individuals (n = 26) than when paGnt.s in the overlap group between type I and @pe I! .s~hizo~hrenia (n = 18) urere compared to normal individu~l,s f~ < O.OOl]. This d~~er~n~e rerna~n~d significant (F < 0.01) peahenoverlap parients were ~~rnp~tr~d to those of type If .s~~li~a~~lrenia. ~ur~~lertn~~re, ~s~h~~~jp~lrgtl~~ patients with lower sc’rum N-ChM proved lo be belter, r~sp[~nder~s fo neurolepri~ therapy. We suggesr that rhesr ~Jl~~~i~te~~ xtwm N-CAM fcvefs reflect an kreased synaptic turnover rate in this pyvchotic sfafe.
Introduction Despite much research, the causes underlying schizophrenia remain obscure. The recent classification of schizophrenia into two syndromes (Crow 1980), type I and type II, has been a productive source of new hypotheses on the etiology of this disorder (Shelley and Walsh 1985). Type I schizophrenia is characterized by positive symptoms (delusions, hallucinations, and thought disorder), which may correlate positively with an increase in brain dopamine receptors (Owen et al. 1985). Type II schizophrenia is associated with negative features (loss or absence of affect, poverty of speech, and loss of volition), which are more likely to be associated with intellectual impairment and structural changes in the brain, as shown by computed tomography and postmortem investigation (Crow et al. 1981). These two syndromes are not exclusive, and overlap may occur in 40% of patients with schizophrenia. The possible role of altered neuronal plasticity, mediated by the formation and degeneration of synapses (Cotman and Nieto-Sampedro 1982, 1984) in the etiology of schizophrenia has only recently been considered (Haracz 1985). An example of this is
770
BIOL PSYCHIATRb 1988:23:769-?I5
postlesional plasticity, where compensatory reinnervation of uninjured afferenth or cprouting results in functional synapses (Nieto-Sampedro et al. 1982). There are over 30 studies of the use of computed tomography in schizophrenia. The finding of enlarged ventricles in most of these suggests a subgroup with neuronal cell loss (Johnstone 1985). This group most resembles Crow’s type II. Of further interest is the finding of increased density of catecholaminergic nerve terminals in some brain areas of patients who had schiz,ophrenia, and this hyperinnervation has been suggested to result from a primary lesion nearby (Hokfeldt et al. 1974; Coyle and Johnston 1980). Here, we have explored the pobstbility of increased synapse turnover being associated with schizophrenia by using 11 \e-rum fragment of the neural cell adhesion molecule (N-CAM). N-CAM is a developmentally regulated cell surface sialoglycoprotein that IS believed to be integrally involved in the structuring and final elaboration of the central nervous system (Jorgensen 198 I; Meier et al. 1984; Choung and Edelman 1984; Edelman 1986). The protein is a complex of three immunologically related polypeptides of 200. 140, and 115 kD molecular weight (Choung and Edelman 1984; Sheehan et al. 1986). Cell adhesion is believed to be mediated by a homophilic binding between N-CAMS on apposing cells (Brackenbury et al. 1977). The strength of this adhesion is dependent on the amount of sialic acid on each molecule (Hoffman and Edelman 1983), and as this decreases during development (Jorgensen 198 1; Rothbard et al. 1982; Choung and Edelman 1984: Meier et al. 1984), increased adhesivity occurs and eventual histogenesis. Recent studies have suggested that in addition to its developmental function. N-CAM may also play a role in the regeneration and restructuring of the mature neuromuscular junction (Covault and Sanes 1985. 1986; Rieger et al. 1985: Daniloff et al. 1986). It is also reasonable to assume that such events occur centrally, as here the neuronal circuitry is being constantly remodeled (Cotman and Nieto-Sampedro 1982, 1984; Nieto-Sampedro et al, 1982). Given that N-CAM has been demonstrated to be sloughed from the cell surface (Thiery et al. 1977; Annunziata et al. 1983), and that this soluble form has also been shown to be present in cerebrospinal fluid and serum (Jorgensen and Bock 1975: Ibsen et al. 1983), several workers have attempted to correlate changes in ~&ublc NCAM expression with altered states of synaptic function in psychotic and neurodegenerative conditions of the central nervous system (Jorgensen and Bock 1975; Jorgensen et al. 1977, 1980, 1982; Soelberg Sorensen et al. 1983). Although no significant correlations were found in these studies, this can be related to the low levels (<4 pgiml) of N-CAM found in human serum and cerebrospinal fluid (Ibsen et al. 1983) and which cannot be readily detected using crossed immunoelectrophoresis (Jorgensen and Bock 1975). Thus, we have considered it worthwhile to reappraise this situation using a sensitive cnzymelinked immunosorbent assay to compare soluble N-CAM levels in the serum (~1schizo-phrenic and control populations.
Methods Patients (26 men, 14 women) were drawn consecutively from a general hospitai clinic (St. James’ Hospital, Dublin) of a community-orientated psychiatric service that they attended for regular depot neuroleptic medication. Forty patients met the inclusion standards of fulfilling Feighner’s criteria (Feighner et al. 1972) for schizophrenia, were aged 18-65 years, gave valid consent, and had no concurrent physical illness. Twenty-six controls (7 men, 19 women) were selected from physically and psychologically normal hospital staff. The mean patient age was 36 years ( f 10.1) and that of the control group
N-CAM Serum Fragment
and Negative Symptoms
BIOL PSYCHIATRY 1988;23:769-775
111
was 33 years ( + i 1.2). The difference between these means is not statistically significant (Student’s 1 = 1.34, df = 64). The patient group was classified into type I (positive symptoms only), type II (negative symptoms only), or overlap type I/Ii (both positive and negative symptoms). The definition of positive and negative subtypes followed was that of Andreasen and Olsen (1982). Only 4 patients could be classified as type I; agreement could not be reached on 5, and in the case of 2 of these, this was because of associated alcohol abuse and mental handicap. Finally, a clinical estimate was made of whether or not patients could be deemed to have responded well to neuroleptic medication. Fourteen were thought to have a good therapeutic outcome, 21 a poor outcome, leaving 5 on whom agreement as to outcome could not be reached. Blood samples were obtained by v~nipuncture and were allowed to clot before the serum was prepared by centrifugation at 3000 rpm for 10 min. N-CAM levels were estimated blindly, using an enzyme-linked immunosorbent assay. Briefly, serum samples were diluted 1:40 in Dulbecco’s phosphate-buffered saline (DPBS), and 50-p] aliquots were allowed to absorb to flat-bottom wells of microtiter plates (A/S Nunc, Roskilde, Denmark) for 18 hr at 4°C. These were then washed 3 times with DPBS containing 0.3% Tween-20, (Sigma Chemical Co., St. Louis, MO), and 50~1 aliquots of an N-CAMspecific antibody (Sheehan et al. 1986), diluted I:20 with DPBS containing 0.05%’ Tween20, were added to each well and incubated for 90 min at room temperature. The wells were again washed and then incubated with 50-pl aliquots of a peroxidase-conjugated sheep anti-rabbit IgG,A, and M (Serotec Ltd.), diluted 1:250 with DPBS containing 0.05% Tween-20. The wells were again washed, and SO-*1 aliquots of Sorensen’s buffer (50 rnM Na2HP04, 25 mM citric acid, pH 5.0), containing the peroxidase substrate ophenylenediamine (0.4 mgiml; Sigma) and HzOz (0.5 pgiml; Sigma) were added to each well. The reaction was allowed to proceed for 5 min before the color was enhanced by the addition of 100 ~1 of 1N H,SO,. The color was read at 492 nm, and the results were expressed in arbitrary units (AU), 1 AU being equivalent to an optical density unit/ microgram protein. Protein concentrations were estimated according to the procedure of Lowry et al. (195 l), and no difference in serum protein content was noted between normal and psychotic individuals. Statistical differences were estimated using an unpaired Student’s r-test. The molecuiar weight of the serum N-CAM fragment was determined using previously described immunoblo~ing procedures with an N-CAM-specific polyclonal antibody (She&n et al. 1986).
Resultts Immunoblotting procedures demonstrated serum N-CAM to comprise a 70-kD fragment, and no evidence of other immunoreactive fragments was obvious (Figure 1). Schizophrenic patients were found to have highly significant increases (p < 0.0001) in serum N-CAM levels when compared to normal individuals (Table l), and this could not be related to differences in age or sex. This difference was more marked between type II schizophrenics and normal individuals (p < O.OOOi) than that of patients in the overlap type I/II group and normal individuals fp < 0.001) (Table 1). Thus, patients with only negative features have higher N-CAM serum levels than patients with mixed positive and negative symptoms. This difference also remained significant (p < 0.01) when overlap patients were compared with those of type II schizophrenia (Table 1). When patients were classified
112
BIOL PSYCHIATK) 1988;23:769-775
Figure I. lmmunoblot demonstrating the molecular weight of the N-CAM serum fragment. The sample, containing SO p,g protein, was separated on a 10% acrylamide gel.
Table I. Serum N-CAM Levels in Schizophrenics and Normal individuals Schizophrenics
N0lTIKTl All
Population size Serum N-CAM units Significance
26
40
x.7 rt 0.22
10.01 to. 16
p ( ~.~~
Values are expresseda%arbitrary unit\ and nn! the mean % XSI.
Type l/II
---__I Type
11
Table 2. Relationship
173
BIOL PSYCHIATRY 1988:23:769-775
N-CAM Serum Fragment and Negative Symptoms
between Serum N-CAM Levels and Therapeutic
Response Response Poor
GOOd
?I
14
Population size Serum N-CAM
units
IO.4
9.1 2 0.18
+ 0.23
Values are expressedas mean * SEMand are significantlydifferent (0.02 > p > 0.01).
according to clinical response to neuroleptic medication, those with a poor therapeutic outcome had significantly higher serum levels (p < 0.02) (Table 2).
Discussion The serum fragment of N-CAM was demonstrated to be 70 kD in molecular weight, and this is very similar to the 6.5kD fragment reported to be generated by spontaneous proteolysis of purified N-CAM (Hoffman et al. 1982). Furthermore, this fragment contains all the antigenic determinants that neutralize the cell adhesion blocking activity of antibodies prepared to intact N-CAM (Hoffman et al. 1982). Consequently, this serum fragment may represent the functional amino-terminal binding domain of the molecule, which would be involved in synapse formation, and an increased release of this could therefore be associated with increased synapse turnover. A possible explanation for elevated levels of N-CAM in the sera of schizophrenics is that neuroleptic therapy may damage neuronal and/or muscle membranes or directly interfere with the enzyme-linked immunosorbent assay (ELBA) solid-phase immunoassay. However, no change in serum N-CAM levels was noted in rats 2 weeks after a depot injection of flupenthixol decanoate or fluphenazine decanoate at twice the concentration given to the patients, and a tenfold excess of therapeutic serum neuroleptic levels (Seeman 1977) was found to have no effect on the ELBA immunoassay procedure (data not shown). Furthermore, in a small but representative group of motorneuron disease patients (n = S), no increase in serum N-CAM was evident (data not shown), suggesting that elevated levels of this protein do not reflect neurodegenerative changes. These results indicate that N-CAM levels can distinguish between a group of normal individuals and patients with a diagnosis of schizophrenia and also between subtypes of this psychotic disorder. However, they must be interpreted with caution. Although there is evidence that N-CAM is involved in synaptic st~cturing and reorganization (Covauft and Sanes 198.5, 1986; Rieger et al. 1983, it is not clear that this would result in an increased release of soluble N-CAM, as would be reflected in serum levels. if this proves to be the case, then natural synaptic turnover rate (Cotman and Nieto-Sampedro 1982, 1984) must be increased and sustained in schizophrenia, and markedly so in type 11 schizophrenia. We gratefully
appreciate the assistance of Mr. Roddy Monks in the preparation of this manuscript.
References Andreasen 794.
NC, Olsen S (1982): Negative v. positive schizophrenia.
Arch Gen ~~~~~~zjff~~35:789-
Annuntiata P, Regan CM. Balazs R f 1983): Development of cerebetlar celis in neuron-enriched cultures: Cell surface proteins. Dev Brain Res 8:2&i-273.
774
6lOL PSYCHIATRY 1988;23:769-77s
Brackenbury R. Thiery J-P. Rutishauser U. Edelman GM (1977): Adhesion among neu;$ CC& ol the chick embryo. I. An immunologi~af assay for molecules involved in ccli-ceit tvnding. .: Rid Ckm 252:68X-6840. Chuong C-C. Edelman GM (1984): Alterations in neural ccl1 adhesion molecuics dtic,itg deuct opment of different regions of the nervous qystcm. J h’eumsci 4:2X4--2368. M C19821: Brain function. synapse renewal and plastictr!
Cotman CW, Nieto-Sampcdro Psyhol 3337 I-401
Cotman CW. Nicto-Sampedro
.‘~wI~KPI,
M I IYX4): Cell biology ot synaptic plasticrty. .Sc,rr,rc,!j.:I’< 17x7.
120-l Coyle JT. Johnston MV ( 1980): Functional hyperinnervation of cerebral cortex by noradrcaergic neurons results from fetal lesions: Parallels with schizophrenia. Psyhophtharmnd 8rtll 16:1779.
Covault J. Sanzs JR ( 1985): Neural celi adhesion ~ll~)le~ule (N-CAM) accumulates and paralyzed skeletal muscles. Proc~ NLII~At& Sci USA 82:4544454X. Covauh J. Sanes JR (1986): Distribution of N-CAM in synaptic and extrasynaptic developing and adult skeletal muscle, .I Cell Bio/ 102:716-730. Crow TJ ( 1980): Molecular J 280:66-6X. Crow TJ. Corsellis
pathology
of schizophrenia:
JAN, Crash AJ. Frith CD, Johnstone
rn ~~f~er~~~t~d portions or
More than one disease concept:
Nt- Mt4
EC. Owen F. Bloxharn C, f.crrlcc- IN.
Owens DGC ( 198 I 1: The search for changes underlying the type II syndrome in schizophrenia. In Penis C. Struwe G. Jansson B (cds). Bio/qiccrl Psyhintp. Amsterdam: Elsevier!North.
Holland Biomedical
Press, pp 727-.73 I
Danilof’f JK. Levi G, Grumet M, Kieger F, Edelman GM (1986): Altered el;presblc,ri ut neurona: cdl adhesion molecules induced by nerve injury and repair. .I Crib Bkrl 103:929-945 Edelman GM ( 1986): Cell adhesion molecules in the regulation of animal form and tisstrc patrcrn. A~7~~~r Ret. Cull Bid 2:81-l 16. Feighner JP, Robins E, Guze SB, Woodruff RF, Winokur G, Munor R f 1973): &agnostic for use in psychiatric research, Awh Get? Psychirzt~ 76:X---63. Haracz JL ( 1985): Neuraf plasticity
in schizophrenia.
trlteria
Schir-ophr B&i I I : 101-X!(3.
Hoffman S, Sorkin BC, Brackenbury R, Mailhammer R, Rutishauser U, Cunningham Bc’. Edelman GM (1982): Chemical characterisation of a neural cell adhesion molecule purified from embryonic membranes. J Bid Chem 257:7720-7729. Hoffman S, Edelman GM (1983): Kinetics of homophilic binding by embryonic and adult iorms of the neural cell adhesion molecule. Proc NatI Ad Sci USA 80:5762--5766. Hokfeldt T, Ljundahl A, Fuxe K. Johansson 0 (1974): Dopamine nerve terminals in the rat hmbic cortex: Aspects of the dopamine hypothesis of schizophrenia. Science 184:177-- 1%. Ibsen S. Berczin V, Norga~d-Pedersen 13. Bock E ( 19831:enzyme-linke~i of the D2-gly~oprotein . J ~~~~~~~~~2~~~ 4 I :356-X2. Johnstone
EC (1985): Structural changes
cho~pharmarnlogy:
Xecrnr
Adwnws
in the brain in schizophrenia. Prospecfs.Oxford:
and Futurr
iI~rnun~~s~~rb~~~~ assay
In lversen SD ted,, PI.!Oxford Medicai Publica-
tions, pp 196-203. Jorgensen OS ( 198 I ): Neuronal 37:939-946.
membrane
DZ-protein during rat brain ontogeny.
.I .~‘~uro&rrr
Jorgensen OS, Bock E (I 975): Synaptic plasma membrane antigen 02 measured in hUmin cercbrospinal fluid by rocket immunoelectrophoresis. Determination in psychiatric and neurological patients. &and .I immunol 425-30. Jorgensen
OS.
cerebrospinal
Bock
E.
Beth
P.
Rafi~elsen
Huid of manic-melancholic
Jorgensen OS, Hemmingsen
synaptic turnover 61:3.56-X4.
OJ (1977): Synaptic membrane protern D2 In the patients. Aeia Ps~chiutr &WZ~ 56:%-56.
R, Kramp P, Rafaefsen OJ (1980): Bled-bran barrier selectivity and during delirium tremens and related clinical states. Artu P.s!‘r*hinrr &XZ~
N-CAM
Serum Fragment
and Ncgativc
775
BIOL PSYCHlrZTRY 1988;1.1m-775
Symptoms
Jorgensen OS, Reynolds GP. Riederer P, Jellinger K ( 1982):Parkinson’s disease putamen: Normal concentration of synaptic membrane marker antigens. J ~eu~~il ~ru~.~~j.~~~54: 17 l-l 79. Lowry OH, Rosebrough NJ. Farr AL, reagent. J Bid Chem 193:X15-275. Meier E. Regan CM. during development
Randall
RJ (1951):
Protein
measurement
with
the Folin
Balazs R (1984): Change in the expression of a neuronal surface protein of cerebellar neuroncs in vivo and in culture. J Neuroc&~m 43: 132X-I 335.
Nicto-Sampedro M. Hoff SF. Cotman CW ( 1982): Perforated synaptic densities: mcdiatcs in synapse turnover. Proc Nnrl Autd Sci USA 7957 18-5722.
Probable
intcr-
Owen F. Crawley J. Cross AJ. Crow TJ. Oldland SR. Poulter M. Veal1 N, Zanelli GD (I985): Dopamine D2 receptors and schizophrenia. In lversen SD (ed). P.~~c,hophnrrnrlc,cti~~~~~: Recw~r Ad~wrzc~~strrzd F~tt~rc Prospec~t.c. Oxford: Oxford Medical Publications. pp 2 16-227 Ricgcr F. Grumet M, Edeiman Crli Biof 101:285-293.
GM (1985):
N-CAM
at the vertebrate
neuromuscular
junction.
.I
Rothbard JB. Brackenbury R. Cunningham BA. Edelman GM t 1982): Differences in the carbohydrate structures of neural ccl1 adhesion molecules from adult and embryonic chicken brains. J Hrc~i C/tern 257: I I .064-I I .Ohl). Sccman P ( 1977): Anti-schizophrenic 26:1741-1748.
drugs. Membrane receptor sites
ofaction. Bid7m7
Plrtrrmc7u~l
Sheehan MC. Halpin CI. Rcgan CM. Moran NM, Kilty CG (1986): Purification and characterization of the D2 cell adhesion protein. Analysis of the postnatally regulated polymorphic forms and their cellular distribution. Ne~~ochrm Res I I : 1333-l 346. Shelley RK. Walsh N (1985): The btochemistry 133.
of schizophrenia-A
review.
Irish Mrd J 7X: 139-
Soelberp Sorensen P. Gjerris F, lbscn S. Bock E (19X3): Low cercbrospinal fluid c~)ncentrati~)n of brain-specific protein D2 in patients with normal pressure hydrocephalus. J ~‘~~~~~~~ SKY6X965. Thicry J-P, Brackenbury R. Rutishauscr U. Edelman GM (1977): Adhesion among ncurai cells of the chick embryo. Ii. Puritication and characterization of a cell adhesion molecule from neural retina. J Biol Chrrn 252:6X31--6X35.