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Novel Neuropeptide Y Processing in Human Cerebrospinal Fluid From Depressed Patients
Peptides,Vol.17,No.7,pp.1107-1111,1996 Copyright 01996 ElsevierScienceInc. Printed in the USA. All rightsreserved 0196-9781/96 $15.00+ .00 ELSEVIER
PII S0196-9781(96)00168-4
Novel Neuropeptide Y Processing in Human Cerebrospinal Fluid From Depressed Patients ROLF EKMAN,* PETER JUHASZ,~l
MARKUS
HEILIG,* HANS ~GREN*
AND CATHERINE
E. COSTELLO~2
*Institute of Clinical Neuroscience, Department of Psychiatry and Neurochemistry, University of Goteborg, Molndals Sjukhus, S-431 80 A401ndal, Sweden fkfass Spectrometry Resource, Department of Chemistry, Massachusetts Institute of Technology,
Cambridgej MA 02139, USA Received 25 March 1996 EKMAN,R., P. JUHASZ,M. HEILIG,H. ikGRENAND C. E. COSTELLO.Novel neuropeptide Yprocessing in human cerebrospirralfiuidfrom depressed patients. PEPTIDES17(7) 1107–1111,1996.—Invitro processingof neuropeptideY (NPY) in cerebrospinalfluid (CSF) of patients with depressionwas monitoredby matrix-assistedlaser desorption/ionizationtime-of-flight mass spectrometry(MALDI-TOFMS). Singlepeptidebondsin NPYwere cleavedto yieldN- or C-terminalfragments.Multiple cleavageto forminternalpeptideswasunimportant.Degradationrates variedbetweenindividuals,whereastheproductdistributions were fairly constant. Other peptides did not evidence such proteolysis.MALDI-TOFMS will facilitate extensiveinvestigations of NPY processing that could provide the basis for clinical assays and illuminate the pathophysiologyrelated to depression. Copyright 01996 Elsevier Science Inc. NeuropeptideY
Depression
MALDI-TOFMS
Humancerebrospinalfluid
NEUROPEPTIDE Y (NPY), a 36-residue peptide with a C-terminal amide (Scheme 1) was first isolated from porcine brain (6,7,23). It is one of the most abundant peptides of the mammalian central nervous system (CNS ) ( 12). By acting through hypothalamic mechanisms, central NPY controls the release of several pituitary hormones and is an important stimulus for ingestion of food, in particular, carbohydrates ( 18,20). In addition, NPY is a major candidate for controlling emotionality by actions in the amygdaloid complex ( 11). A Y] subtype of NPY receptors has been cloned. It belongs to the family of G-protein-coupled receptors and requires the intact NPY sequence for activation. A Y2 population, characterized pharmacologically, can also be activated by truncated C-terminal NPY fragments. Additional subtypes of NPY receptors are likely to exist ( 10). Thus, processing of the NPY molecule represents a powerful mechanism for modulating its biological actions by preferentially activating selected receptor subpopulations. It has, however, not yet been possible, with conventional analytical methods, to obtain complete peptide processing patterns. The biosynthesis of neuropeptides starts from large preprohormones; these undergo several transformation steps during biomaturation to the active peptide in response to various stimuli before being degraded and inactivated ( 19). Cerebrospinal fluid (CSF) reflects metabolic events related to normal homeostasis of the CNS as well as pathophysiological conditions. A neuropep-
YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY-NH2 neuropeptideY HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 glucagon-likepeptide(7-36)amide RPKPQQFFGLM-NH2
substanceP
AGCKNFFWKTFTSC
somatostatin
RPVKVYPNGAEDESAEAFPLEF
ACTH(18-39)
SCHEME1. Structuresof peptideswhose stability in human CSF was investigatedin vitro under the incubationconditionsdescribed.
tide fragment profile in CSF reflects to some extent the metabolic balance between different cell types of the brain and the spinal cord. Decreased levels of NPY-like immunoreactivity (NPY-LI) have been reported in the CSF of human subjects with major depression, compared to sex and age matched controls (24). However, others have failed to observe changes in CSF levels of NPY-LI among depressed patients (4). These divergent results may result from an altered processing of NPY into different immunoreactive fragments with different bioactivity, rather than a change in the overall synthesis.
‘ Present address: Perceptive Biosystems,Framingham,MA 01701. 2Requests for reprints shouldbe addressedto Ca;herineE. Costello at her present address, Departmentof Biophysics,Boston UniversitySchool of Medicine,80 E, ConcordStreet, R-806,Boston,MA 02118-2394.
1107
1108
EKMAN ET AL.
High performance liquid chromatography (HPLC), in combination with radioimmunoassay (RIA), has been used to follow NPY processing (25), but the results from this technique are affected by the specificity of the antibody and thus yield only an incomplete picture of the processing and chemical modifications of the neuropeptide. The approach has obvious limitations for obtaining accurate information on the metabolic perturbations of neuropeptides. Mass spectromettic techniques have long played an important role in the anrdysis of biological materials. Characterization of neuropeptides in human CSF using fast atom bombardment mass spectrometry, for fractions separated by HPLC, has been reported (9,17). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) ( 13,16,22) is a relatively new technique and has been demonstrated to be a powerful analytical method for studying peptides and proteins. The strengths of MALDI-TOF MS include high sensitivity (picomole to low femtomole) and the ability to examine complex samples without requirement for extensive purification. The problem-solving potential of this technique for neurochemical studies has been demonstrated recently (5,14). It can be used without any sample pretreatment to study directly the in vitro processing of peptides in biological fluids, reflecting in vivo conditions during different pathologic conditions. We report here the utilization of MALDITOF MS to follow NPY processing in vitro in CSF from depressed patients. For this study, CSF samples were obtained from five patients with DSM-111-Rdiagnoses of major depression (Table 1) and an age-matched healthy control. Patients formed part of a sample of 58 individuals collected in a depression research protocol. The sample of five was representative of the larger sample in terms of subdiagnoses and symptom ratings as shown by nonsignificant statistical tests of means. As a general rule, patients had a washout period for antidepressant medication for at least 2 weeks prior to the lumbar puncture. METHOD
CSF Samples Lumbar punctures were performed in a standardized manner in the lateral decubitus position in the L4–L5 vertebral interstice, following rules reported earlier ( 1). The 12-ml CSF samples were centrifuged at 2000 X g for 10 min to remove cells and other insoluble material and stored at –80”C. CSF samples ( 100 pl) were mixed with phosphate buffer (usually 10 mkf, pH 7) and lyophilized before shipping from Sweden to the US. In each
u 1-36
1-32
.30
1-34
mlz
1000
1600
2200
2800
3400
4000
FIG. 1.MALDI-TOFmass spectnrmof a depressedpatient’sCSFspiked with a high level (25 pmol/pl) of neuropeptideY, after incubationfor 6 h (10 mkf buffer, pH 7). 2,5-Dihydroxybenzoicacid matrix.Numerical intervalsabovethe peaksindicateaminoacid residuesretainedfrom the originalsequence.Mass assignmentsare given in Table 2. experiment, the lyophilized sample was reconstituted with water (usually to 100 pl) and the solution was spiked with 1-10 pl of NPY solution in water (0.2–10 pmol/~1) in a l-ml Eppendorf tube. The tubes were incubated at 37°C for 18 or 42 h. For MALDI-TOF MS analysis at various time points, 1-w1aliquots were withdrawn. Each aliquot was mixed with 5–10 volumes of the matrix solution (see below), and 1 pl of the resulting mixture was applied to the target. MALDI-TOF MS MALDI-TOF MS measurements were carried out on a VT2000 (Vestec Corp., now Perceptive Biosystems, Framingham, MA) 2-m linear time-of-flight mass spectrometer. The instrument, data acquisition, and data processing are described in detail elsewhere (15 ). A nitrogen laser (Laser Science Inc., Newton, MA) radiating at 337 nm was used for desorptionlionization. Ions were accelerated to 30 keV. External calibration was used to assign the major peaks; these were then used as standards for internal calibration to complete the mass assignments. To obtain a comprehensive picture of the neuropeptide processing in MALDI experiments, three matrices were used: sinapinic acid (SA) (2), 2,5-dihydroxybenzoic acid (2,5 -DHB) (21), and a-cyano-4-hydroxycinnamic acid (a-CHCA) (3). During peptide anrdysis, each matrix shows some discrimination. SA was prepared as a saturated solution in 2: 1 water/acetonitrile
TABLE 1 CLINICAL CHARACTERISTICS OF PATIENTS WITHDSM-111-R MAJORDEPRESSION
No.
sex
Age
Subdiagnosis
Concomitant Diagnosis
363 369 376 382 395
M M F M F
44 54 39 40 36
Primaryunipolar BipolarII Primaryunipolar Primaryunipolar Secondaryunipolar
— — — BorderlinePD
Psychosis
xHRDS Worst Week
xHRDS Past Week
GAS Worst Week
GAS Past Week
no no no no no
24 22 25 23 25
24 19 25 13 25
35 35 50 40 30
35 55 50 65 30
xHRDS:HamiltonDepressionRating Scale extractedfrom the Scheduleof AffectiveDisordersand Schizophrenia(SADS)[J. Endicott,J. Cohen, J. Fleiss, S. Sarantakos(1981).Hamiltondepressionrating scale: extractedfrom regular and changeversionsof the Schedulefor AffectiveDisorders of Schizophrenia,Archivesof General Psychiatry,38, 98– 103].Worst Week: Ratings duringthe most severely depressedweek during the present affect episode.Past Week: Ratingsduringthe week precedinglumbarpuncture.GAS: GlobalAssessmentScale includedin the SADS.
1109
NEUROPEPTIDE Y PROCESSING IN HUMAN CSF TABLE 2 CALCULATED AND OBSERVED mlz VALUES FOR (M + H)+ OF NPY AND ITS TRUNCATED FRAGMENTS Amino Acid Residues
1-8 (OH)
1-16(OH) 1-17(OH) 1-18(OH) 1-21( OH) 1-22( OH) 1-23(OH) 1-24(OH) 1–25(OH) 1-26(OH) 1-27(OH) 1-28(OH) 1-29(OH) 1-30(OH) 1-32(OH) 1-34(OH) 1–36(NH2)
mlz Calc. *
m/z Obs. T
917.0 1701.7 1832.9 1904.0 2386,6 2473.6 2544.7 2657.9 2814.1 2951.2 3114.4 3227.5 3341.7 3454.8 3669.1 3953.4 4272,8
917.2 2703.4 1835.6 1906.0 2388.0 2474.9 2546.2 2659.2$ 2814.3 2951.3 3114.2 3224.9 3341.6 3453.0 3669.9 3953.5 4272.5*
Amino Acid Residues
mlz Calc. *
m/z Obs.i-
34-36 (NH,) 33-36(NH, ) 32-36(NH2) 31-36(NH,) 30-36(NH,) 29-36(NH,) 28-36(NH,) 27–36(NH,) 26–36(NH2)
465,2 621.3 722.8 836.0 949.1 1063.3 1176.4 1339.6 1476,7
467.0 621.0 721.7 836.1 949.3 1063,2$ 1176.9 1340,1 1475.5
1-36(NH2)
4272.8
4272.5*
* Averagemass values. 1’Data are from the mass spectrumshownin Fig. 1. ~ Peak identifiedon the basis of external calibrationand subsequentlyused as interual standardfor mass assignment.
have a C-terminal carboxyl group or free N-terminus at the cleavage site and are not further degraded. To verify that the incubation conditions alone were not responsible for the observed degradation of NPY, some experiments were conducted over wide concentration ranges of both substrate (NPY, 2– 100 pmol/pl CSF) and buffer (phosphate, pH 7, 5– 100 mkf). It was found that the relative abundance distribution of products varied somewhat with concentration, indicating the existence of multiple processing pathways or the loss of kinetic control over substrate site selectivity at high concentrations, and suggesting the possibility of surface effects at very low concentrations. In buffered saline solution or at high buffer levels in the presence of CSF, only negligible decomposition
(W/A); a-CHCA was dissolved at 10 g/1 in 1:1 W/A, 2,5-DHB at 10 g/1 in water. RESULTS
From the MALDI-TOF mass spectra, such as that shown in Fig. 1, it is evident that extensive processing of NPY occurs during incubation with CSF from some individuals. Degradation takes place via cleavage of one of the C—N bonds along the peptide backbone to produce C- and N-terminally truncated analogues of the initial peptide (Table 2). Formation of internal fragments is insignificant. The newly formed shortened peptides 100
T (a)
50
0
‘-
2+
100
P--=7+4==-~
# $ j
d
r
H 100-
(c)
m
50
0 ml.
50
? .. $ ; .
“-—7—’
0
--— ]700
2200
2?(1U
3200
3700
4200
FIG. 2. MALDI-TOFmass spectra acquiredfor aliquotsof a solutionof neuropeptideY, incubatedat differentbufferconcentrations,taken at the 24-h time point: (a) 50 mM buffer, pH 7; (b) 10mM buffer, pH 7; (c) pure water. a-Cyano-4-hydroxycinnamicacid matrix.
mlz
1400
2000
2600
3200
3800
4400
FIG, 3. MALDI-TOFmass spectra acquired for a depressed patient’s CSF samplespikedwith neuropeptideY (5 pmol/pl, 10mM buffer,pH 7), taken at sequentialtime points as indicatedto the left of each trace. Sinapinicacid matrix.
1110
EKMAN ET AL.
occurred. At low buffer levels, decomposition occurred, but the extent and pattern were different from that observed in the presence of CSF. The results of varying buffer concentration are illustrated in Fig. 2. The question whether the extensive decomposition of NPY upon incubation with CSF is a unique characteristic of this peptide rather than a general phenomenon was investigated by subjecting to the experimental conditions four other peptides whose structures are shown in Scheme 1. The neuropeptides substance P and glucagon-like peptide(7–36) amide (GLP), which resemble NPY in having C-terminal amides, exhibited very low amounts of N- or C-terminal truncation; the cyclic peptide somatostatin and ACTH( 18–39), similar in size to NPY but having a C-terminal carboxyl group, were resistant to proteolysis under the same conditions (data not shown). The relative abundance pattern of the NPY degradation products does not vary as a function of time, as demonstrated in Fig. 3, wherein are overlaid the spectra acquired for a single depressed patient’s CSF sample after different incubation times. Although the extent of conversion of NPY to products increases with time, and the overall level of products rises, the relative abundance distribution of products appears to be quite the same throughout the process. When CSF samples from different patients and a control are compared [Fig. 4(a) control, (b) patients], it becomes clear that the overall distribution pattern is fairly constant but the rates of processing vary strongly among patients and differ from the control. Additional control samples have been examined in the MIT laboratory during the initial phase of this study and more recently in the Goteborg laboratory; none of the control samples has shown the extensive degradation behavior observed for patient samples. Because opportunities for obtaining CSF samples are limited by clinical needs, it has not yet been possible to track the pattern and rate for a single subject sampled at different times.
W.
2200
3200
2700
4200
3700
100
I 1-36
(b) 1-24
1-29
: :
27
4,0. ..? % ~
1-32 /.l)l”l
CSF 395 CSF 382 ..— CSF 376
0
CSF 363
m/z
I
I
I
I
I
I
2200
2700
3200
3700
4200
4700
/
FIG. 4. MALDI-TOFmass spectraacquiredfor six differentindividuals’ humanCSF samplesspikedwith nenropeptideY (2.5 pmol/@, 10 mM buffer, pH 7), taken after 18-hincubation.(a) Normalcontrol.(b) Five patients whose clinical states are described in Table 1. Sinapinicacid matrix.
Y
Y-P-S-K-P-D-N-P-G-E-D-A-P-A-E-D-M-A1 2
3 4 5
6
11
7 8
9 10 1112 13 141516 17 18
R-Y-Y-S-A-L-R-H-Y-l-N-L19 202122
232425
J
I-T: R-Q-R-Y-NH2
26 27282930313233343536
SCHEME2. Aminoacid sequenceof neuropeptideY markedto indicate favored scissionpoints duringincubationin humanCSF. Relative sizes of arrowsindicateincreasingproclivityfor cleavageat the site marked. DISCUSSION Neuropeptides are known to play important roles in brain function, as neurotransmitters and neuromodulators, and are to some extent involved in the complex regulation of different processes such as cognition, memory, and behavior. Although the role of the peptides in cellular communication/regulation is not well understood, they represent the most diverse class of transmitter molecules. The measurement of neuropeptides in CSF represents one of the first steps in attempting to link chemical perturbations in peptidergic neurons with psychopathology. RIA of neuropeptides in the CSF is complicated by the fact that neuropeptides display considerable complexity through chemical modifications produced during maturation and metabolism (8). It is necessary, therefore, to have available an analytical method that has sensitivity comparable to RIA yet exhibits molecular specificity. In the experiments described here MALDI-TOF MS was shown to meet these criteria, and to provide new information about NPY degradation pathways. For all the human CSF samples analyzed under the conditions selected, certain positions were found to be favored for proteolysis (Scheme 2). No single type of amino acid residue is preferred exclusively. This behavior suggests that the cleavage sites are controlled by the topology of enzyme-substrate interactions. It should be usefhl to study how changes in amino acid residue(s) affect this behavior, to determine whether certain residues or subsequences are critical. Such information may give insight as to the compositional or steric requirements for cleavage. When the biological activities (if any) of the truncated peptides have been determined, the intriguing question of whether there is a pharmacological advantage or disadvantage to increased decomposition rate may be answered. Processing of the NPY molecule into fragments with differential biological activities represents a potential substrate for pathophysiological mechanisms in psychiatric disease states. It has, for example, been demonstrated that endogeneous NPY acts to reduce anxiety by activating Y1 receptors in the amygdala, whereas truncated NPY analognes lack this ability (11). The MALDI-TOF MS methodology described in this report provides a sensitive and rapid means to address these issues. The approach is one that can be readily adapted for processing large numbers of samples. The examinationof a larger pool of samples from patients with variousdiagnoseswill show whether there is aclirtical comelation with the inherent degradation rate variations. ACKNOWLEDGEMENTS Theautbomaregratefulto K.Biemarrnforhelpfoldiscussionsandto J. E. Billerfor providingspecializedsoftware.The MIT Mass SpectmmetryResourcewas supportedby ND-INationalCenterfor ResearchResourcesGrant
NEUROPEPTIDE Y PROCESSING IN HUMAN CSF
1111
No.RRO0317 (to K. Bie). TheSwedishMcdicrdResearchCouncilprovidcda travelgrantto R. Ekmarrand supportathe researchat the University
of G6teborg.ThisstudywasapprovedbytheEthicalCommitteeof G&eborg Universityandthepatientsgavetheirinformedconsent.
REFERENCES H.; Mefford,N.; Rudmfer,M. V.; Linnoila,M.; Pr.rtter,W. Z. Interacting neurotransmittersystems. A nonexperimentalapproach to the 5 HIAA-HVAcorrelationin human CSF. J. Psychiatr.Res. 20:175-193; 1986. 2. Beavis R. C.; Chait, B. T. Cirnmmicacid derivativesas matricesfor ultraviolet laser resorption mass spectrometryof proteins. Rapid Commun.Mass Specrrom.3:432–435; 1989. 3. Bemis, R. C.; Chaudhary,T.; Chait, B. T. a-Cyano-4-hydroxycinnamic acid as a matrix for matrix-assisted laser resorption mass spectrometry.Org. Mass Spectrom.27:156–158; 1992. 4. Berrettini,W. H.; Duran,A. R.; Kelwe, J.; Roy,A.; Pickar,D. Cerebrospinal fluid neuropeptideY in depression and schizophrenia. Neuropsychopharmacology1:81-83; 1987. 5. Chou,J. Z.; Kreek,M. J.; Chait, B. T. Matrix-assistedlaser resorption mass spectrometryof biotransformationproductsof dynorphin A in vitro. J. Am. Soc. Mass Spectrom.5:10-16; 1993. 6. Dawbarn,D.; Hunt, S. P.; Emson,P. C. NeuropeptideY: Regional distribution, chromatographiccharacterization and imrnunohistochemical demonstrationin postmortem human brain. Brain Res. 296:168-173; 1984. 7. DeQuill, M. E.; Emson, P. C. Distributionof neuropeptideY-like immumrreactivityin the rat centraf nervous system—I. Radioimmunoassay and chromatographic characterisation. Neuroscience 18:527-543; 1986. 8. Ekman,R. Radioimmunoassayof neuropeptidesin the CSF. Problems and pitfalls. Nerd. Psykiatr.Tidskr. 39:31-34; 1985. 9. Giamsta, E.-L.; Nyberg,F.; Silberring,J. Applicationof fast-atom bombardmentmass spectrometryfor sequencingof a hemoglobin fragment, naturally occurring in human cerebrospinalfluid. Rapid Commun.Mass Spectrom.6:777-780; 1992. 10. Grundemar,L.; Sheikh, S. P.; Wahlestedt, C. Characterizationof receptortypes for nerrropeptideY and related peptides.In: Colmers, W. F.; Wahlestedt,C., eds. The biologyof neuropeptideY. Totowa, NJ: HumanaPress; 1993:197–240. 11. Heilig, M.; Koob, G. F.; Ekman, R.; Britton, K. T. Corticotropinreleasing factor and neuropeptideY: Role in emotionalintegration. Trends Neurosci. 17:80–85; 1994. 12. Hendry,S. H. C. Organizationof NPYneuronsin CNS.In: Colmers, W. F.; Wahlestedt,C., eds. The biologyof neuropeptideY. Totowa, NJ: HumanaPress; 1993:65-156. 13. Hillenkamp,F.; Karas, M. Mass spectrometryof peptides and proteins by matrix-assistedultraviolet laser desorption/ionization.In: McCloskey,J., ed. Methods in enzymology,vol. 193.Mass spectrometry.San Diego,CA: AcademicPress; 1990:280–295. 1. &ren,
14. Jim6nez,C. R.; van Veelen, P. A.; Li, K. W.; et al, Neuropeptide expressionand processingas revealedby direct matrix-assistedlaser resorption ionizationmass spectrometryof single neurons. J. Neurochem.62:404–407;1994. 15. Juhasz, P.; Costello, C. E.; Biemarm,K. Matrix-assistedlaser resorption ionization mass spectrometry with 2-(4-hydroxyphenylazo)benzoicacid matrix.J. Am.Soc.Mass Spectrom.4:399–409; 1993. 16. Karas,M.; Bachmann,D,; Bahr, U.; Hillenkamp,F. Matrix-assisted ultravioletlaser resorption of nonvolatilecompounds.Int. J. Mass Spectrom.Ion Processes78:53-68; 1987. 17. Liu, D.; Dass, C.; Wood,G.; Desiderio,D. M. Opiodand tacbykinin peptides and their precursors and precursor-processingenzymesin humanCSF.J. Chromatogr.500:395;1990, 18. McDonald,J. K.; Koenig,J. I. NeuropeptideY actionson reproductive and endocrinefunctions, In: Colmers, W. F.; Wahlestedt, C., eds. The biology of neuropeptideY. Totowa, NJ: Humana Press; 1993:419-456. 19. Minth, C. D.; Bloom, S. R.; Polak, J, M,; Dixon, J. E. Cloning, characterization,andDNAsequenceof humancDNAencodingneuropeptide tyrosine. Proc. Natl. Acad. Sci. USA 81:4577–4581; 1984. 20. Stanley, B. G. Neuropeptide Y in multiple hypothalamic sites controls eating, behavior, endocrine and autonomic systems for
21. 22. 23. 24.
25.
body energy balance. In: Colmers, W. F.; Wahlestedt, C., eds. The biology of neuropeptide Y. Totowa, NJ: Humana Press; 1993:457-510. Strupat, K.; Karas, M.; Hillenkamp, F. 2.5-Dihydroxybenzoic acid: A new matrix for laser desorption-ionization mass spectrometry. Int. J. Mass Spectrom. Ion Processes 111:89–102; 1991. Tanaka, K.; W&i, H.; Ido, Y.; Akita, S. Protein and polymer analyses up to tiz 100,000 by laser ionization time-of-flight mass spectrometry. Rapid Commrm. Mass Spectrom. 3:151–153; 1989. Tatemoto, K.; Carlquist, M.; Mutt, V. Neuropeptide Y—a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296:659–660; 1982. Widerlov, E.; Lindstrom, L. H.; Wahlestedt, C.; Ekman, R. Neuropeptide Y (NPY) and peptide YY (PYY) as possible cerebrospinal fluid markers for major depression and schizophrenia, respectively. J. Psychiatr. Res. 22:69-79; 1988. Widerlov, E.; Heilig, M.; Ekman, R.; Wahlestedt, C. Neuropeptide Y—possible involvement in depression and anxiety. In Mutt, V., et al., eds. Neuropeptide Y. New York: Raven Press; 1989:331– 342.
PII S0196-9781(96)00168-4
Novel Neuropeptide Y Processing in Human Cerebrospinal Fluid From Depressed Patients ROLF EKMAN,* PETER JUHASZ,~l
MARKUS
HEILIG,* HANS ~GREN*
AND CATHERINE
E. COSTELLO~2
*Institute of Clinical Neuroscience, Department of Psychiatry and Neurochemistry, University of Goteborg, Molndals Sjukhus, S-431 80 A401ndal, Sweden fkfass Spectrometry Resource, Department of Chemistry, Massachusetts Institute of Technology,
Cambridgej MA 02139, USA Received 25 March 1996 EKMAN,R., P. JUHASZ,M. HEILIG,H. ikGRENAND C. E. COSTELLO.Novel neuropeptide Yprocessing in human cerebrospirralfiuidfrom depressed patients. PEPTIDES17(7) 1107–1111,1996.—Invitro processingof neuropeptideY (NPY) in cerebrospinalfluid (CSF) of patients with depressionwas monitoredby matrix-assistedlaser desorption/ionizationtime-of-flight mass spectrometry(MALDI-TOFMS). Singlepeptidebondsin NPYwere cleavedto yieldN- or C-terminalfragments.Multiple cleavageto forminternalpeptideswasunimportant.Degradationrates variedbetweenindividuals,whereastheproductdistributions were fairly constant. Other peptides did not evidence such proteolysis.MALDI-TOFMS will facilitate extensiveinvestigations of NPY processing that could provide the basis for clinical assays and illuminate the pathophysiologyrelated to depression. Copyright 01996 Elsevier Science Inc. NeuropeptideY
Depression
MALDI-TOFMS
Humancerebrospinalfluid
NEUROPEPTIDE Y (NPY), a 36-residue peptide with a C-terminal amide (Scheme 1) was first isolated from porcine brain (6,7,23). It is one of the most abundant peptides of the mammalian central nervous system (CNS ) ( 12). By acting through hypothalamic mechanisms, central NPY controls the release of several pituitary hormones and is an important stimulus for ingestion of food, in particular, carbohydrates ( 18,20). In addition, NPY is a major candidate for controlling emotionality by actions in the amygdaloid complex ( 11). A Y] subtype of NPY receptors has been cloned. It belongs to the family of G-protein-coupled receptors and requires the intact NPY sequence for activation. A Y2 population, characterized pharmacologically, can also be activated by truncated C-terminal NPY fragments. Additional subtypes of NPY receptors are likely to exist ( 10). Thus, processing of the NPY molecule represents a powerful mechanism for modulating its biological actions by preferentially activating selected receptor subpopulations. It has, however, not yet been possible, with conventional analytical methods, to obtain complete peptide processing patterns. The biosynthesis of neuropeptides starts from large preprohormones; these undergo several transformation steps during biomaturation to the active peptide in response to various stimuli before being degraded and inactivated ( 19). Cerebrospinal fluid (CSF) reflects metabolic events related to normal homeostasis of the CNS as well as pathophysiological conditions. A neuropep-
YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY-NH2 neuropeptideY HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 glucagon-likepeptide(7-36)amide RPKPQQFFGLM-NH2
substanceP
AGCKNFFWKTFTSC
somatostatin
RPVKVYPNGAEDESAEAFPLEF
ACTH(18-39)
SCHEME1. Structuresof peptideswhose stability in human CSF was investigatedin vitro under the incubationconditionsdescribed.
tide fragment profile in CSF reflects to some extent the metabolic balance between different cell types of the brain and the spinal cord. Decreased levels of NPY-like immunoreactivity (NPY-LI) have been reported in the CSF of human subjects with major depression, compared to sex and age matched controls (24). However, others have failed to observe changes in CSF levels of NPY-LI among depressed patients (4). These divergent results may result from an altered processing of NPY into different immunoreactive fragments with different bioactivity, rather than a change in the overall synthesis.
‘ Present address: Perceptive Biosystems,Framingham,MA 01701. 2Requests for reprints shouldbe addressedto Ca;herineE. Costello at her present address, Departmentof Biophysics,Boston UniversitySchool of Medicine,80 E, ConcordStreet, R-806,Boston,MA 02118-2394.
1107
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EKMAN ET AL.
High performance liquid chromatography (HPLC), in combination with radioimmunoassay (RIA), has been used to follow NPY processing (25), but the results from this technique are affected by the specificity of the antibody and thus yield only an incomplete picture of the processing and chemical modifications of the neuropeptide. The approach has obvious limitations for obtaining accurate information on the metabolic perturbations of neuropeptides. Mass spectromettic techniques have long played an important role in the anrdysis of biological materials. Characterization of neuropeptides in human CSF using fast atom bombardment mass spectrometry, for fractions separated by HPLC, has been reported (9,17). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) ( 13,16,22) is a relatively new technique and has been demonstrated to be a powerful analytical method for studying peptides and proteins. The strengths of MALDI-TOF MS include high sensitivity (picomole to low femtomole) and the ability to examine complex samples without requirement for extensive purification. The problem-solving potential of this technique for neurochemical studies has been demonstrated recently (5,14). It can be used without any sample pretreatment to study directly the in vitro processing of peptides in biological fluids, reflecting in vivo conditions during different pathologic conditions. We report here the utilization of MALDITOF MS to follow NPY processing in vitro in CSF from depressed patients. For this study, CSF samples were obtained from five patients with DSM-111-Rdiagnoses of major depression (Table 1) and an age-matched healthy control. Patients formed part of a sample of 58 individuals collected in a depression research protocol. The sample of five was representative of the larger sample in terms of subdiagnoses and symptom ratings as shown by nonsignificant statistical tests of means. As a general rule, patients had a washout period for antidepressant medication for at least 2 weeks prior to the lumbar puncture. METHOD
CSF Samples Lumbar punctures were performed in a standardized manner in the lateral decubitus position in the L4–L5 vertebral interstice, following rules reported earlier ( 1). The 12-ml CSF samples were centrifuged at 2000 X g for 10 min to remove cells and other insoluble material and stored at –80”C. CSF samples ( 100 pl) were mixed with phosphate buffer (usually 10 mkf, pH 7) and lyophilized before shipping from Sweden to the US. In each
u 1-36
1-32
.30
1-34
mlz
1000
1600
2200
2800
3400
4000
FIG. 1.MALDI-TOFmass spectnrmof a depressedpatient’sCSFspiked with a high level (25 pmol/pl) of neuropeptideY, after incubationfor 6 h (10 mkf buffer, pH 7). 2,5-Dihydroxybenzoicacid matrix.Numerical intervalsabovethe peaksindicateaminoacid residuesretainedfrom the originalsequence.Mass assignmentsare given in Table 2. experiment, the lyophilized sample was reconstituted with water (usually to 100 pl) and the solution was spiked with 1-10 pl of NPY solution in water (0.2–10 pmol/~1) in a l-ml Eppendorf tube. The tubes were incubated at 37°C for 18 or 42 h. For MALDI-TOF MS analysis at various time points, 1-w1aliquots were withdrawn. Each aliquot was mixed with 5–10 volumes of the matrix solution (see below), and 1 pl of the resulting mixture was applied to the target. MALDI-TOF MS MALDI-TOF MS measurements were carried out on a VT2000 (Vestec Corp., now Perceptive Biosystems, Framingham, MA) 2-m linear time-of-flight mass spectrometer. The instrument, data acquisition, and data processing are described in detail elsewhere (15 ). A nitrogen laser (Laser Science Inc., Newton, MA) radiating at 337 nm was used for desorptionlionization. Ions were accelerated to 30 keV. External calibration was used to assign the major peaks; these were then used as standards for internal calibration to complete the mass assignments. To obtain a comprehensive picture of the neuropeptide processing in MALDI experiments, three matrices were used: sinapinic acid (SA) (2), 2,5-dihydroxybenzoic acid (2,5 -DHB) (21), and a-cyano-4-hydroxycinnamic acid (a-CHCA) (3). During peptide anrdysis, each matrix shows some discrimination. SA was prepared as a saturated solution in 2: 1 water/acetonitrile
TABLE 1 CLINICAL CHARACTERISTICS OF PATIENTS WITHDSM-111-R MAJORDEPRESSION
No.
sex
Age
Subdiagnosis
Concomitant Diagnosis
363 369 376 382 395
M M F M F
44 54 39 40 36
Primaryunipolar BipolarII Primaryunipolar Primaryunipolar Secondaryunipolar
— — — BorderlinePD
Psychosis
xHRDS Worst Week
xHRDS Past Week
GAS Worst Week
GAS Past Week
no no no no no
24 22 25 23 25
24 19 25 13 25
35 35 50 40 30
35 55 50 65 30
xHRDS:HamiltonDepressionRating Scale extractedfrom the Scheduleof AffectiveDisordersand Schizophrenia(SADS)[J. Endicott,J. Cohen, J. Fleiss, S. Sarantakos(1981).Hamiltondepressionrating scale: extractedfrom regular and changeversionsof the Schedulefor AffectiveDisorders of Schizophrenia,Archivesof General Psychiatry,38, 98– 103].Worst Week: Ratings duringthe most severely depressedweek during the present affect episode.Past Week: Ratingsduringthe week precedinglumbarpuncture.GAS: GlobalAssessmentScale includedin the SADS.
1109
NEUROPEPTIDE Y PROCESSING IN HUMAN CSF TABLE 2 CALCULATED AND OBSERVED mlz VALUES FOR (M + H)+ OF NPY AND ITS TRUNCATED FRAGMENTS Amino Acid Residues
1-8 (OH)
1-16(OH) 1-17(OH) 1-18(OH) 1-21( OH) 1-22( OH) 1-23(OH) 1-24(OH) 1–25(OH) 1-26(OH) 1-27(OH) 1-28(OH) 1-29(OH) 1-30(OH) 1-32(OH) 1-34(OH) 1–36(NH2)
mlz Calc. *
m/z Obs. T
917.0 1701.7 1832.9 1904.0 2386,6 2473.6 2544.7 2657.9 2814.1 2951.2 3114.4 3227.5 3341.7 3454.8 3669.1 3953.4 4272,8
917.2 2703.4 1835.6 1906.0 2388.0 2474.9 2546.2 2659.2$ 2814.3 2951.3 3114.2 3224.9 3341.6 3453.0 3669.9 3953.5 4272.5*
Amino Acid Residues
mlz Calc. *
m/z Obs.i-
34-36 (NH,) 33-36(NH, ) 32-36(NH2) 31-36(NH,) 30-36(NH,) 29-36(NH,) 28-36(NH,) 27–36(NH,) 26–36(NH2)
465,2 621.3 722.8 836.0 949.1 1063.3 1176.4 1339.6 1476,7
467.0 621.0 721.7 836.1 949.3 1063,2$ 1176.9 1340,1 1475.5
1-36(NH2)
4272.8
4272.5*
* Averagemass values. 1’Data are from the mass spectrumshownin Fig. 1. ~ Peak identifiedon the basis of external calibrationand subsequentlyused as interual standardfor mass assignment.
have a C-terminal carboxyl group or free N-terminus at the cleavage site and are not further degraded. To verify that the incubation conditions alone were not responsible for the observed degradation of NPY, some experiments were conducted over wide concentration ranges of both substrate (NPY, 2– 100 pmol/pl CSF) and buffer (phosphate, pH 7, 5– 100 mkf). It was found that the relative abundance distribution of products varied somewhat with concentration, indicating the existence of multiple processing pathways or the loss of kinetic control over substrate site selectivity at high concentrations, and suggesting the possibility of surface effects at very low concentrations. In buffered saline solution or at high buffer levels in the presence of CSF, only negligible decomposition
(W/A); a-CHCA was dissolved at 10 g/1 in 1:1 W/A, 2,5-DHB at 10 g/1 in water. RESULTS
From the MALDI-TOF mass spectra, such as that shown in Fig. 1, it is evident that extensive processing of NPY occurs during incubation with CSF from some individuals. Degradation takes place via cleavage of one of the C—N bonds along the peptide backbone to produce C- and N-terminally truncated analogues of the initial peptide (Table 2). Formation of internal fragments is insignificant. The newly formed shortened peptides 100
T (a)
50
0
‘-
2+
100
P--=7+4==-~
# $ j
d
r
H 100-
(c)
m
50
0 ml.
50
? .. $ ; .
“-—7—’
0
--— ]700
2200
2?(1U
3200
3700
4200
FIG. 2. MALDI-TOFmass spectra acquiredfor aliquotsof a solutionof neuropeptideY, incubatedat differentbufferconcentrations,taken at the 24-h time point: (a) 50 mM buffer, pH 7; (b) 10mM buffer, pH 7; (c) pure water. a-Cyano-4-hydroxycinnamicacid matrix.
mlz
1400
2000
2600
3200
3800
4400
FIG, 3. MALDI-TOFmass spectra acquired for a depressed patient’s CSF samplespikedwith neuropeptideY (5 pmol/pl, 10mM buffer,pH 7), taken at sequentialtime points as indicatedto the left of each trace. Sinapinicacid matrix.
1110
EKMAN ET AL.
occurred. At low buffer levels, decomposition occurred, but the extent and pattern were different from that observed in the presence of CSF. The results of varying buffer concentration are illustrated in Fig. 2. The question whether the extensive decomposition of NPY upon incubation with CSF is a unique characteristic of this peptide rather than a general phenomenon was investigated by subjecting to the experimental conditions four other peptides whose structures are shown in Scheme 1. The neuropeptides substance P and glucagon-like peptide(7–36) amide (GLP), which resemble NPY in having C-terminal amides, exhibited very low amounts of N- or C-terminal truncation; the cyclic peptide somatostatin and ACTH( 18–39), similar in size to NPY but having a C-terminal carboxyl group, were resistant to proteolysis under the same conditions (data not shown). The relative abundance pattern of the NPY degradation products does not vary as a function of time, as demonstrated in Fig. 3, wherein are overlaid the spectra acquired for a single depressed patient’s CSF sample after different incubation times. Although the extent of conversion of NPY to products increases with time, and the overall level of products rises, the relative abundance distribution of products appears to be quite the same throughout the process. When CSF samples from different patients and a control are compared [Fig. 4(a) control, (b) patients], it becomes clear that the overall distribution pattern is fairly constant but the rates of processing vary strongly among patients and differ from the control. Additional control samples have been examined in the MIT laboratory during the initial phase of this study and more recently in the Goteborg laboratory; none of the control samples has shown the extensive degradation behavior observed for patient samples. Because opportunities for obtaining CSF samples are limited by clinical needs, it has not yet been possible to track the pattern and rate for a single subject sampled at different times.
W.
2200
3200
2700
4200
3700
100
I 1-36
(b) 1-24
1-29
: :
27
4,0. ..? % ~
1-32 /.l)l”l
CSF 395 CSF 382 ..— CSF 376
0
CSF 363
m/z
I
I
I
I
I
I
2200
2700
3200
3700
4200
4700
/
FIG. 4. MALDI-TOFmass spectraacquiredfor six differentindividuals’ humanCSF samplesspikedwith nenropeptideY (2.5 pmol/@, 10 mM buffer, pH 7), taken after 18-hincubation.(a) Normalcontrol.(b) Five patients whose clinical states are described in Table 1. Sinapinicacid matrix.
Y
Y-P-S-K-P-D-N-P-G-E-D-A-P-A-E-D-M-A1 2
3 4 5
6
11
7 8
9 10 1112 13 141516 17 18
R-Y-Y-S-A-L-R-H-Y-l-N-L19 202122
232425
J
I-T: R-Q-R-Y-NH2
26 27282930313233343536
SCHEME2. Aminoacid sequenceof neuropeptideY markedto indicate favored scissionpoints duringincubationin humanCSF. Relative sizes of arrowsindicateincreasingproclivityfor cleavageat the site marked. DISCUSSION Neuropeptides are known to play important roles in brain function, as neurotransmitters and neuromodulators, and are to some extent involved in the complex regulation of different processes such as cognition, memory, and behavior. Although the role of the peptides in cellular communication/regulation is not well understood, they represent the most diverse class of transmitter molecules. The measurement of neuropeptides in CSF represents one of the first steps in attempting to link chemical perturbations in peptidergic neurons with psychopathology. RIA of neuropeptides in the CSF is complicated by the fact that neuropeptides display considerable complexity through chemical modifications produced during maturation and metabolism (8). It is necessary, therefore, to have available an analytical method that has sensitivity comparable to RIA yet exhibits molecular specificity. In the experiments described here MALDI-TOF MS was shown to meet these criteria, and to provide new information about NPY degradation pathways. For all the human CSF samples analyzed under the conditions selected, certain positions were found to be favored for proteolysis (Scheme 2). No single type of amino acid residue is preferred exclusively. This behavior suggests that the cleavage sites are controlled by the topology of enzyme-substrate interactions. It should be usefhl to study how changes in amino acid residue(s) affect this behavior, to determine whether certain residues or subsequences are critical. Such information may give insight as to the compositional or steric requirements for cleavage. When the biological activities (if any) of the truncated peptides have been determined, the intriguing question of whether there is a pharmacological advantage or disadvantage to increased decomposition rate may be answered. Processing of the NPY molecule into fragments with differential biological activities represents a potential substrate for pathophysiological mechanisms in psychiatric disease states. It has, for example, been demonstrated that endogeneous NPY acts to reduce anxiety by activating Y1 receptors in the amygdala, whereas truncated NPY analognes lack this ability (11). The MALDI-TOF MS methodology described in this report provides a sensitive and rapid means to address these issues. The approach is one that can be readily adapted for processing large numbers of samples. The examinationof a larger pool of samples from patients with variousdiagnoseswill show whether there is aclirtical comelation with the inherent degradation rate variations. ACKNOWLEDGEMENTS Theautbomaregratefulto K.Biemarrnforhelpfoldiscussionsandto J. E. Billerfor providingspecializedsoftware.The MIT Mass SpectmmetryResourcewas supportedby ND-INationalCenterfor ResearchResourcesGrant
NEUROPEPTIDE Y PROCESSING IN HUMAN CSF
1111
No.RRO0317 (to K. Bie). TheSwedishMcdicrdResearchCouncilprovidcda travelgrantto R. Ekmarrand supportathe researchat the University
of G6teborg.ThisstudywasapprovedbytheEthicalCommitteeof G&eborg Universityandthepatientsgavetheirinformedconsent.
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