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Original Studies |
Graduate School Neurosciences, Netherlands Institute for Brain Research, Amsterdam, The Netherlands; Clinical Research Institute of Montreal (N.G.S.), Montreal, Canada; Laboratory for Molecular Oncology, Center for Human Genetics, Flanders Interuniversity Institute for Biotechnology, University of Leuven (J.W.V.d.L., W.J.M.V.d.V.), Leuven, Belgium; and the Department of Animal Physiology, University of Nijmegen (G.J.M.M.), Nijmegen, The Netherlands
Address all correspondence and requests for reprints to: Dr. B. A. Th. F. Gabreëls, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam ZO, The Netherlands. E-mail: B.Gabreels{at}nih.knaw.nl
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Introduction |
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The current study was initiated to investigate the expression of the major neuroendocrine peptides VP and OT in the SON and PVN (14), in particular in those two PWS patients that had a disturbed expression of 7B2. Therefore, we tested immunocytochemically the expression of different parts of the VP precursor and OT in the SON and PVN of seven PWS hypothalami. Because of the discrepancy found in the two 7B2-negative PWS patients, viz.; no staining of processed VP, whereas antibodies against other parts of the VP precursor revealed positive staining, we investigated the possibility of a processing defect by determining the expression of the neuroendocrine convertases PC1 and PC2 in these hypothalamic cells.
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Materials and Methods |
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) were obtained at autopsy. In addition,
the hypothalami of 7 clinically diagnosed PWS patients were studied
(for details of 5 of the 7 PWS patients, see Refs. 4 and 9), the
clinical data of the other two new patients are given below). The
control patients were in the same range for age, postmortem delay, and
fixation time. The required separate permission for brain autopsy was
obtained either from the patients themselves or their partners or
relatives. Neuropathological investigation of the control subjects was
performed by Drs. W. Kamphorst (Free University, Amsterdam, The
Netherlands), D. Troost (Academic Medical Center, Amsterdam, The
Netherlands), or Prof. F. C. Stam (Netherlands Brain Bank,
Amsterdam, The Netherlands). The hypothalamus was dissected out and
fixed in 4% formaldehyde at room temperature. Subsequently, tissue was
routinely dehydrated and embedded in paraffin. Serial 6-µm coronal
sections were cut and mounted on chrome-alum-coated glass slides.
Sections for microwave treatment were mounted on Superfrost/Plus glass
slides (Menzel-Gläser, Braunschweig, Germany).
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The patient was a 25-yr-old woman. Pregnancy was uncomplicated. Nothing was reported about the quantity or quality of fetal movements by the mother. The patient was a floppy infant with a weak cry and was not able to suck during the first days of life. The patient became obese at the age of 2 yr and from that time had been kept on an energy-reduced diet (1200 Cal/day). She took food from everywhere, and the kitchen had to be locked. Her face was "typical for PWS," and her hands and feet were small. Pubarche occurred at 13 yr of age. She showed some mammary development but no menarche at 16 yr (last hospital records). At the age of 15 yr, the patient was able to read, but not to calculate. No task problems could be solved without guidance and support. There was no diabetes mellitus, and the glucose tolerance test was normal. Hypothyroidism was found and was treated with T4 (0.05–0.1 mg/day). Other hormone levels were: FSH, 19 IE/L (normal levels, 2–30 IU); and LH, above 2 IE/L (normal levels, 3–120 IE/L depending on the stage of the menstrual cycle). There was a normal GH response on glucagon stimulation, a normal computed tomography scan and x-ray of the cranium, and no abnormalities on the electroencephalogram. At autopsy she had a length of 155 cm, and a weight of 52 kg. The uterus and ovaries were of normal size and shape. For one marker (D15S97), the patient showed a deletion in the PWS region. She had 9 points according to the diagnostic criteria of Holm (15), with at least 8 points being necessary for a positive PWS diagnosis.
Case 6: 95-104 (Mental Retardation Institute, Zwammerdam, The Netherlands)
The patient was a 51-yr-old man. There was no history of hypotonia or poor suckling reflex during the neonatal period. At the age of 5 yr he developed obesity. The patient had a "characteristic face" for PWS. In adulthood, a small penis and a left testis with a volume of 9 mL were reported. The right testis was removed curatively because of a seminoma at the age of 29 yr. There was a normal sexual hair pattern. His length was originally 1.65 m, but was reduced later on by scoliosis; his weight was 92 kg. Small hands and feet were observed. The patient was mentally retarded, with an IQ of 49, and exhibited a number of behavioral problems, such as temper fits, obstinate behavior, stealing food, and nicotine addiction. There was no apneu syndrome or rapid eye movement sleep disturbances, but there was an abnormal hypnogram, showing very little deep sleep, too many arousals, and excessive sleepiness during the day. He also developed hypertension and hypercholesterolemia without diabetes mellitus. Endocrinological investigations showed no thyroid or adrenal abnormalities or disturbances of the X-sella turcica. He probably died of cardiac arrhythmia from a 3-week-old infarct of the left chamber of the heart complicated by bronchopneumonia of the right lung. According to the diagnostic criteria of Holm et al. (15), the patient had 8.5 points of the required 8 for PWS diagnosis. Molecular genetic analysis revealed no deletion, but maternal disomy was shown with probe PW71, which is indicative of PWS.
Immunocytochemistry
For immunocytochemistry, deparaffinized sections were incubated with the following antibodies (for details of the procedure, see Ref.9).
VP antibodies. Monoclonal mouse anti-VP (VP III-D-7) was used, which recognizes Phe in position 3 as the most important determinant in the VP ring, diluted 1:200 (provided by Dr. A. Hou-Yu, Columbia University, New York, NY) (16). In the nonprocessing cell line, HEK-293, transfected with the human VP gene, there was no reaction with this antibody. However, in the Neuro2A cell line (17), which shows regulated pathway processing, transfected with the human VP gene an intense reaction was obtained, indicating that only processed VP was recognized.
Polyclonal rabbit anti-VP preabsorbed with OT was used; it preferentially recognizes VP in its processed form (Truus, 29–01-1986; diluted 1:1000) (18).
Neurophysin (NP) antibodies. Monoclonal mouse anti-NP, with the most likely epitope located between amino acids 75 and 76 of the NP moiety, was used (19) (PS41, diluted 1:200, provided by Dr. H. Gainer, NIH, Bethesda, MD) (20). In immunoprecipitation assays the antibody brought down NP as well as its precursor molecule synthesized in vivo, suggesting that this antibody recognizes both the VP precursor and processed NP (20).
Polyclonal rabbit anti-NP directed against a synthetic human N-terminal NP fragment representing the residues 1–12 plus Tyr (N-Term NP, diluted 1:500, prepared by Dr. A. G. Robinson, University of California-Los Angeles and supplied by Pituitary Hormones and Antisera Center, Director A.F. Parlow). In tricine SDS-PAGE according to Schägger (21) and subsequently Western blotting, the antibody recognizes predominantly processed NP.
Polyclonal rabbit anti-NP directed against a synthetic human C-terminal NP fragment of the residues 80–91 plus Tyr was used (C-Term NP, diluted 1:500, prepared by Dr. A. G. Robinson, University of California-Los Angeles and supplied by Pituitary Hormones and Antisera Center, Bethesda, MD) (22). In tricine SDS-PAGE and subsequently Western blotting, the antibody recognizes predominantly processed NP.
Glycopeptide (GP) antibodies. Polyclonal rabbit anti-GP, against a synthetic human GP-(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39) [Boris, diluted 1:1000, provided by W. G. North, Dartmouth Medical School (Lebanon, NH)] (23). This antibody reacts in both the HEK-293 and Neuro2A (17) cell lines, so it at least recognizes the VP precursor. In tricine SDS-PAGE and subsequently Western blotting, the antibody recognizes predominantly the VP precursor.
Polyclonal rabbit anti-GP directed against the guinea pig glycoprotein moiety of the VP precursor was used [K 1.7, diluted 1:500, provided by I.C.A.F. Robinson, National Institute for Medical Research (London, UK)] (24). This antibody reacts in both the HEK-293 and Neuro2A (17) cell lines, which indicates that it at least recognizes the VP precursor. In tricine SDS-PAGE and subsequently Western blotting, the antibody recognizes predominantly the VP precursor.
OT antibody. Monoclonal mouse anti-OT, with three different antigenic determinants on the OT molecule was used: the Ile in position 3, Pro in 7, and Leu in 8 [A-I-28, diluted 1:200, provided by Dr. A. Hou-Yu, Columbia University (New York, NY)] (25).
7B2 antibodies. Polyclonal rabbit anti-7B2 directed against synthetic human 7B2 23–39 was used (RB-7, diluted 1:500) (26).
Monoclonal mouse anti-human 7B2, recognizing 7B2 sequence 64–94, was used [MON-144 (supernatant), diluted 1:10, provided by H. L. P. van Duynhoven (Helmond, The Netherlands)] (27).
Monoclonal mouse anti-human 7B2, recognizing 7B2 sequence 128–143, was used [MON-102 (ascites), diluted 1:1000, provided by H. L. P. van Duynhoven] (27).
Polyclonal rabbit anti-mouse 7B2 directed directed against residues 156–186 and recognizing 156–171 of the C-terminus of 7B2 was used (CT 7B2, Rb-4, 14-2-1991, diluted 1:250) (28).
PC1 antibodies. Polyclonal rabbit anti-PC1 directed against human PC1 fragment 43–628 was used (PC1, diluted 1:500). Specificity in the Western blot assay was positive for AtT20 cell lysate and negative for bacterial fusion proteins with parts of PC2, PC4, PACE4, PC6A, the C-terminus of PC6B, and the prodomain of furin. It was positive in immunopre cipitation [transfected PK (15) cells, no reaction in untransfected cells], immunofluorescence positive in AtT20 cells as endogenous activity and in transfected cells, and negative in COS-1 cells.
Polyclonal rabbit antimouse PC1 directed against residues 84–100 was used [88% homology with human PC1; 2B6 N-terminal, diluted 1:500, provided by I. Lindberg, Louisiana State University, New Orleans, LA)] (29).
Polyclonal rabbit antimouse PC1 directed against residues 629–726 was used (PC1 92-12-08 C-terminal, diluted 1:500) (30).
PC2 antibody. Polyclonal rabbit anti-human PC2 directed against residues 122–637 was used (PC2, diluted 1:500). Specificity in the Western blot assay was negative for AtT20 cell lysate and bacterial fusion proteins with parts of PC1, PC4, PACE4, PC6A, the C-terminus of PC6B, and the prodomain of furin. It was positive in immunoprecipitation [transfected PK (15) cells, no reaction in untransfected cells], immunofluorescence positive in transfected PK (15) cells, and negative in untransfected PK (15) cells and AtT20 cells.
The intensity of the immunoreactivity was estimated semiquantitatively. The tissue, which stained very faintly or not at all with some antibodies, was applied to microwave pretreatment according to a modified procedure, based on that reported by Shi et al. (31) as described by Lucassen et al. (32), to increase the sensitivity. After deparaffinization, the sections were treated according to the following procedure: 1) a 15-min wash in distilled water; 2) incubation in a microwave oven twice for 5 min each time in a citric acid solution (0.1 mol/L citric acid monohydrate and 0.1 mol/L trisodium citrate dihydrate), pH 6.0; 3) a 2-fold 5-min wash in Tris-buffered saline; and 4) incubation in 5% nonfat dried milk for 30 min (Elk, Campina, Eindhoven, The Netherlands).
To determine whether PC1, PC2, and 7B2 are coexpressed in VP or OT cells, we stained sections of a control subject with VP antibody III-D-7 or OT antibody A-I-28 and the consecutive section with, respectively, 7B2 antibody MON-102, PC1 antibody PC1, and PC2 antibody PC2.
Specificity controls
For specificity of the VP, NP, GP, and OT antibodies, see the procedures and references described above. The antibodies raised against different parts of 7B2 were adsorbed with the recombinant glutathione-S-transferase-7B2 hybrid protein (prepared by D. W. Eib) (26). PC1 and PC2 were absorbed with their respective recombinant antigens PC1 and PC2. 2B6 N-terminal PC1 and PC1 92-12-08 C-terminal were absorbed with their respective antigen residues (2B6 N-terminal PC1 antigen, provided by I. Lindberg, Louisiana State University). After adsorption, no reaction was seen, nor was there any reaction when the first antibodies were omitted from the procedure.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) No clear difference in
staining was observed with regard to age, sex, postmortem delay, or
fixation time. The SON and PVN of the hypothalami of two PWS patients
(92-005 and 93-056) previously shown to be deficient for 7B2 did not
display processed VP immunoreactivity with either III-D-7 (Fig. 1A) or Truus (see Table 1). However,
these patients showed a very intense staining for the processed NP and
GP parts of the VP precursor and the precursor itself, as demonstrated
by the staining with NP antibodies (PS41, N-Term NP, and C-Term NP) and
GP antibodies (Boris and K 1.7) in the SON and PVN (Table 1 and Fig. 1, B and C). The absence of processed VP staining in PWS patient 93-056
was not due to the long fixation time, because a control patient
(95-83) with a much longer fixation time (607 days) had very intense
processed VP immunoreactivity. Two PWS patients (91-058 and 95-104)
showed moderate processed VP immunoreactivity and no MON-102
immunoreactivity, but there was staining with the other 7B2 antibodies,
indicating some degree of VP processing (Tables 1 and 2). To improve the detection level of the
immunocytochemical method, we used the microwave method and found
moderate staining with antibody MON-102 in PWS patient (92-005) and
only faint to moderate 7B2 staining for all four 7B2 antibodies in PWS
patient (93–056; Table 2 and Fig. 2A).
Unfortunately, there was no material available to perform the same
procedure on a PWS patient (92-005) for the other 7B2 antibodies. With
the same procedure, a very intense processed VP staining appeared in
the PWS patient (92-005) and a moderate processed VP staining was found
in PWS patient 93-056 (Fig. 2B). A normal staining intensity for OT was
found in PWS patients compared with that in control patients (Table 1
and Fig. 3).
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, A–D; 5,
A–D; and 6, A–D).
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). PC1 immunoreactivity was present in
all PWS patients and controls, but the intensity was lower for PC1 and
absent for PC1 2B6 N-terminal in the 7B2-nonreacting PWS patients
(92-005 and 93-056). PC2 immunoreactivity was not detectable in the
7B2-nonreacting PWS patients (Fig. 7A),
but all other PWS and control patients showed clear PC2 staining (Fig. 7B). After microwave pretreatment, the PC2-nonreacting PWS patients
(92-005 and 93-056) showed only very faint to faint staining for PC2
(Table 3 and Fig. 7C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main finding of the present study is that the absence of the neuroendocrine polypeptide 7B2 in two PWS patients (92-005 and 93-056) coincides with the absence of processed VP and PC2 in the SON and PVN. In addition, our results showed that PC1 and PC2 are predominantly located in VP cells. In these two PWS patients, however, the VP precursor appeared to be present normally, as shown by antibodies against NP and GP. These observations strongly suggest a processing defect. The absence of 7B2 probably leads to a disturbed pro-PC2 transport from the endoplasmic reticulum to the Golgi complex and subsequently to a lack of activation of PC2. The described results fully agree with the in vitro finding of Braks et al. (10), that 7B2 is a molecular chaperone of pro-PC2. Our results indicate, moreover, that PC2 might be a candidate enzyme for cleaving the dibasic amino acid site between VP and NP in the VP precursor because the absence of PC2 leads to no processed VP.
The very intense (92-005) to moderate (93-056) staining of processed VP, a moderate reaction with 7B2, and faint to very faint PC2 immunoreaction after microwave pretreatment in both PWS patients indicate that although these proteins are expressed to an abnormally low degree, they are not totally absent. We can be confident that the microwave procedure itself does not induce staining, because in another group of patients deficient in processed VP (Wolfram syndrome) using the same procedure, no immunoreactivity for processed VP, 7B2, or PC2 (Gabreëls, B. A. Th. F., submitted for publication) was seen. One can at present only speculate about the mechanism behind the reduced expression of these proteins. It could be that PWS patients 92-005 and 93-056 have such a large deletion on the paternal allele of chromosome 15 that not only the PWS region (15q11–13) but also the near 7B2 gene (15q13–14) or its promotor region are disturbed. This region of chromosome 15 is known to be particularly unstable (37). In agreement with the appearance of some 7B2 immunoreactivity after improving the sensitivity of the staining by microwave treatment is the finding that the 7B2 gene is probably not part of the imprinting region on chromosome 15 (38); thus, the 7B2 allele of the mother in PWS patients (92-005 and 93-056) can still be expressed. Unfortunately, it is impossible at this moment to obtain a molecular genetic diagnosis of paraffin-embedded material to establish the deletion in these PWS patients. The DNA extracted from the cerebellum of one patient (93-056) appeared to be highly fragmented (Smeets, H. J. M., personal communication). In this regard, it is of interest that in patient 95-104, the premortem molecular genetic diagnosis showed maternal disomy, and this patient had no deletion and stained well for 7B2.
Syndromes have been described with inherited defects in processing in an autosomal dominant pattern arising from mutations within the cleavage sites of certain proproteins, such as proinsulin (39) or blood coagulation factor IX (40). In addition, patients have been mentioned in the literature with a normal precursor, viz. POMC (41, 42) or proinsulin (42), but without detectable processing products. Recently, in the patient described by O’Rahilly, a mutation in the PC1 gene was reported (43). However, the present report is the first involving patients showing a disturbance in the regulation of a processing enzyme itself.
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Acknowledgments |
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Received July 9, 1997.
Revised October 8, 1997.
Accepted October 17, 1997.
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