Neuroscience Letters, 139 (1992)47~,9 g" 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/S 05.00
47
NSL 08590
Marked increase in cerebrospinal fluid ubiquitin in Creutzfeldt-Jakob disease H i d e o M a n a k a ~, T a k e o K a t o " , Keiji K u r i t a '~, T a d a s h i K a t a g i r ? , Y u k i h i r o S h i k a m a ~', K a y o k o K u j i r a i ~', T o r u K a w a n a m ? , Y o s h i h i r o S u z u k i ~, K u n i n o b u N i h e i ~, H i d e o S a s a k i ~', S h i g e r u Y a m a d a ~, K o i c h i H i r o t a b, H i r o fumi Kusaka ~ and Terukuni ImaV ~Third Department ql'lnternal Medicbw. Yamagata University School o/ Medicine, )~mul~ata f Japan). ~'Department qlNeurolo%y. Akita Red ('r~s~ Ho~spital. Akita (Japan) amUDepartment o1' Neurolo~,,y. Kilano Hospital. Osaka (Jap~m)
(Received 9 December 1991: Revised version received 7 February 1992: Accepted 10 February 1992 Key words: Ubiquitin; Creutzfeldt Jakob disease: Cerebrospinal fluid: Senile dementia of the Alzheimcr type: M ulti-infarct dementia: Radioimlnu-
noassay We have established the radioimmunoassay lk~rubiquitin in the cerebrospinal fluid (CSF) and measured the ubiquitin concentration in CSF From 4 cases of neuropathologically verified Creutzfeldt Jakob disease (CJD), l0 cases of multi-intarct dementia (MID), 7 cases of senile dementia of Alzheimer type (SDAT), and 18 controls. The normal values were determined to range from 7.3 to 21.0 ng/ml, 14.3 4 I. 1 ng,'mlin the mean + S.E.M. The CSF ubiquitin levels in the cases of MID and SDAT were 16.6 + 6.4 ng/ml and 21.3 ± 6.1 ng/ml, respectively. In the cases of CJD, the ('SF ubiquitin was markedly increased at the early and middle stages of the disease (230.6 ng/ml in Case 1, 107.6 ng/ml m Case 2, 212.5 ng/ml in Case 3, and 377.0 ng/ml in Case 4) and these gradually decreased as the disease progressed. The measurement ofCSF ubiquitin seems useful to make an earl} diagnosis of CJD.
Creutzfeldt J a k o b disease (CJD) [6] is a fatal disease which affects middle-aged and elderly people. The patients show progressive dementia a c c o m p a n i e d by a variety o f s y m p t o m s and signs o f the central nervous system (CNS), and these rapidly worsen, resulting in death usually within a few years. Since this disease is caused by a transmissible agent which has not yet been completely characterized and this agent is highly resistant to ordinary physical or chemical treatments, special warning must be given to medical workers to avoid infection with and spread o f the agent. A l t h o u g h the combination o f progressive dementia, myoclonic jerks, electroencephalographic ( E E G ) findings such as periodic synclonous discharge (PSD), and rapidly progressing brain a t r o p h y on C T or M R I is fairly characteristic o f C J D , these florid manifestations usually appear at the middle or advanced stage o f the disease. The l a b o r a t o r y findings, including blood, urine and cerebrospinal fluid (CSF), are usually normal. A definite diagnosis can be made only by histological examination o f brain-biopsied Curre,wondence." T. Kato, Third Department of Internal Medicine, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-23, Japan.
tissue and its transmissibility to animals. Therefore, it is urgent to establish other non-invasive examinations useful for the diagnosis o f CJD. In this c o m m u n i c a t i o n , we report that ubiquitin, which is a signal peptide for the ATP-dependent non-lysosomal proteolytic system in cells [3], was markedly increased in the C S F of patients with CJD. By modifying the method of Haas and Bright [1], we raised anti-ubiquitin antiserum in rabbits injected with purified ubiquitin (Sigma) coupled to bovine serum albumin (BSA) (Sigma). Ubiquitin was measured by r a d i o i m m u n o a s s a y ( R I A ) using the ubiquitin-specific antiserum at a final dilution of 1:10,500. The assay buffer contained 0.01 M phosphate, 0.14 M NaCI, 0.025 M E D T A , 0.5% BSA and 0.02% NaN~ at a final pH 7.4. Radiolabelled ubiquitin was prepared by the chloramine-T m e t h o d [5]. A mixture o f the buffer, antiserttm, sample and tracer in a final volume o f 0.7 ml was incubated at 4°C for 48 h, and a n t i b o d y - b o u n d and -unb o u n d tracer were separated using anti-rabbit )'-globulin goat serum. The least detectable concentration o f ubiquitin was 1.0 ng/ml, lntra-assay variation was 5% and interassay variation was 10%. C S F was collected from 4 cases of CJD, 10 cases o f multi-infarct dementia (MID),
48
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MONTHS Z
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Fig. 2. The relationship between /he duration of illness (months after onset) and CSF ubiquitin levels in patients with CJD (Cases 1 and 2). CSF ubiquitin levels are markedly elevated at the early and middle stages of CJD and these decrease as the disease progresses. Cases 1, 2, 3 and 4 are ,,,hown by the numbers 1, 2, 3 and 4, respectively. I indicates the point of death.
200
O
100
i__
-:;:
0 CONT
CJD
MID
SDAT
Fig. 1. Scattergram of CSF ubiquitin levels in controls (CONT, n - 181 and in patients with Creutzfeldt.--Jakob disease (CJD, n - 4), multiinfarct dementia (MID, n = 10) and senile dementia of the Alzheimcr type (SDAT, n = 7). Horizontal bars indicate the mean for each group. Methods: anti-ubiquitin antiserum was raised in rabbit against ubiquitin-BSA conjugate and was used for radioimmunoassay at a final dilution of 1:10,500. ~2Sl-ubiquitin was prepared by the chloramine-T method. The least detectable concentration of ubiquitin was 1.0 ng/ml. CSF was stored at - 2 0 ° C until assay.
7 cases of senile dementia of Alzheimer type (SDAT), and 18 patients who initially had headache or dizziness and were later proved to have no organic lesions in the nervous system (controls). The CSF samples were stored at - 2 0 ° C until assay. All CJD cases (Cases 1~1, 54--74 years of age) presented with profound dysfunctions of the CNS including dementia, emotional changes, myoclonus of the extremities and other neurological symptoms and signs, and finally did not respond to any
stimuli within a year. All showed characteristic PSD patterns in EEG and rapidly progressing brain atrophy on repeated MRI or CT scan examinations. The patients were then in a vegetative state and died 4 months to 3 years after the initial symptoms. Postmortem examination of the brains was performed in all four cases and the clinical diagnosis was confirmed by neuropathological examination (panencephalopathic type of CJD in Cases 1 and 2, and spongiform encephalopathy in Cases 3 and 4) [4, 9]. The clinical diagnosis of SDAT was based on the criteria by McKhann et al. [8] and on MR! and/or CT scan findings. The CSF ubiquitin levels of the controls ranged from 7.3 to 21.0 ng/ml, 14.3 z 1.1 ng/ml in the mean 7- S.E M. (Fig. 1). The CSF ubiquitin concentrations in the cases of M I D and SDAT were 16.6 z 6.4 ng/ml and 2t.3 ~- 6.1 ng/ml, respectively (Fig. 1L in the CJD cases, the CSF levels of ubiquitin were markedly elevated ranging from 107.6 to 377.0 ng/ml at the early and middle stages of the disease (230.6 ng/ml in Case 1, 107.6 ng/ml in Case 2. 212.5 ng/ml in Case 3 and 377.0 ng, ml in Case 4) (Fig. 1). No, or only a mild, increase m CSF protein was observed in these cases. In Cases 1 and 2. we could examine CSF samples at several different points of the clinical course and the CSF ubiquitin values were found to decrease gradually as the disease progressed (Fig. 2). Since ubiquitin is required for degradation of abnormal proteins in cells [1, 3], the CSF ubiquitin increase in CJD may be due to its overproduction in CNS in response to noxious agentls), such as prion, and its leakage fi'om CNS to CSF. In this regard, of particular
49
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47
NSL 08590
Marked increase in cerebrospinal fluid ubiquitin in Creutzfeldt-Jakob disease H i d e o M a n a k a ~, T a k e o K a t o " , Keiji K u r i t a '~, T a d a s h i K a t a g i r ? , Y u k i h i r o S h i k a m a ~', K a y o k o K u j i r a i ~', T o r u K a w a n a m ? , Y o s h i h i r o S u z u k i ~, K u n i n o b u N i h e i ~, H i d e o S a s a k i ~', S h i g e r u Y a m a d a ~, K o i c h i H i r o t a b, H i r o fumi Kusaka ~ and Terukuni ImaV ~Third Department ql'lnternal Medicbw. Yamagata University School o/ Medicine, )~mul~ata f Japan). ~'Department qlNeurolo%y. Akita Red ('r~s~ Ho~spital. Akita (Japan) amUDepartment o1' Neurolo~,,y. Kilano Hospital. Osaka (Jap~m)
(Received 9 December 1991: Revised version received 7 February 1992: Accepted 10 February 1992 Key words: Ubiquitin; Creutzfeldt Jakob disease: Cerebrospinal fluid: Senile dementia of the Alzheimcr type: M ulti-infarct dementia: Radioimlnu-
noassay We have established the radioimmunoassay lk~rubiquitin in the cerebrospinal fluid (CSF) and measured the ubiquitin concentration in CSF From 4 cases of neuropathologically verified Creutzfeldt Jakob disease (CJD), l0 cases of multi-intarct dementia (MID), 7 cases of senile dementia of Alzheimer type (SDAT), and 18 controls. The normal values were determined to range from 7.3 to 21.0 ng/ml, 14.3 4 I. 1 ng,'mlin the mean + S.E.M. The CSF ubiquitin levels in the cases of MID and SDAT were 16.6 + 6.4 ng/ml and 21.3 ± 6.1 ng/ml, respectively. In the cases of CJD, the ('SF ubiquitin was markedly increased at the early and middle stages of the disease (230.6 ng/ml in Case 1, 107.6 ng/ml m Case 2, 212.5 ng/ml in Case 3, and 377.0 ng/ml in Case 4) and these gradually decreased as the disease progressed. The measurement ofCSF ubiquitin seems useful to make an earl} diagnosis of CJD.
Creutzfeldt J a k o b disease (CJD) [6] is a fatal disease which affects middle-aged and elderly people. The patients show progressive dementia a c c o m p a n i e d by a variety o f s y m p t o m s and signs o f the central nervous system (CNS), and these rapidly worsen, resulting in death usually within a few years. Since this disease is caused by a transmissible agent which has not yet been completely characterized and this agent is highly resistant to ordinary physical or chemical treatments, special warning must be given to medical workers to avoid infection with and spread o f the agent. A l t h o u g h the combination o f progressive dementia, myoclonic jerks, electroencephalographic ( E E G ) findings such as periodic synclonous discharge (PSD), and rapidly progressing brain a t r o p h y on C T or M R I is fairly characteristic o f C J D , these florid manifestations usually appear at the middle or advanced stage o f the disease. The l a b o r a t o r y findings, including blood, urine and cerebrospinal fluid (CSF), are usually normal. A definite diagnosis can be made only by histological examination o f brain-biopsied Curre,wondence." T. Kato, Third Department of Internal Medicine, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-23, Japan.
tissue and its transmissibility to animals. Therefore, it is urgent to establish other non-invasive examinations useful for the diagnosis o f CJD. In this c o m m u n i c a t i o n , we report that ubiquitin, which is a signal peptide for the ATP-dependent non-lysosomal proteolytic system in cells [3], was markedly increased in the C S F of patients with CJD. By modifying the method of Haas and Bright [1], we raised anti-ubiquitin antiserum in rabbits injected with purified ubiquitin (Sigma) coupled to bovine serum albumin (BSA) (Sigma). Ubiquitin was measured by r a d i o i m m u n o a s s a y ( R I A ) using the ubiquitin-specific antiserum at a final dilution of 1:10,500. The assay buffer contained 0.01 M phosphate, 0.14 M NaCI, 0.025 M E D T A , 0.5% BSA and 0.02% NaN~ at a final pH 7.4. Radiolabelled ubiquitin was prepared by the chloramine-T m e t h o d [5]. A mixture o f the buffer, antiserttm, sample and tracer in a final volume o f 0.7 ml was incubated at 4°C for 48 h, and a n t i b o d y - b o u n d and -unb o u n d tracer were separated using anti-rabbit )'-globulin goat serum. The least detectable concentration o f ubiquitin was 1.0 ng/ml, lntra-assay variation was 5% and interassay variation was 10%. C S F was collected from 4 cases of CJD, 10 cases o f multi-infarct dementia (MID),
48
400
400,
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4
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300
1
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100~
m E
A
m
t-
0
v
12
24
m
B
allm
m
+
t
36
MONTHS Z
~_.
Fig. 2. The relationship between /he duration of illness (months after onset) and CSF ubiquitin levels in patients with CJD (Cases 1 and 2). CSF ubiquitin levels are markedly elevated at the early and middle stages of CJD and these decrease as the disease progresses. Cases 1, 2, 3 and 4 are ,,,hown by the numbers 1, 2, 3 and 4, respectively. I indicates the point of death.
200
O
100
i__
-:;:
0 CONT
CJD
MID
SDAT
Fig. 1. Scattergram of CSF ubiquitin levels in controls (CONT, n - 181 and in patients with Creutzfeldt.--Jakob disease (CJD, n - 4), multiinfarct dementia (MID, n = 10) and senile dementia of the Alzheimcr type (SDAT, n = 7). Horizontal bars indicate the mean for each group. Methods: anti-ubiquitin antiserum was raised in rabbit against ubiquitin-BSA conjugate and was used for radioimmunoassay at a final dilution of 1:10,500. ~2Sl-ubiquitin was prepared by the chloramine-T method. The least detectable concentration of ubiquitin was 1.0 ng/ml. CSF was stored at - 2 0 ° C until assay.
7 cases of senile dementia of Alzheimer type (SDAT), and 18 patients who initially had headache or dizziness and were later proved to have no organic lesions in the nervous system (controls). The CSF samples were stored at - 2 0 ° C until assay. All CJD cases (Cases 1~1, 54--74 years of age) presented with profound dysfunctions of the CNS including dementia, emotional changes, myoclonus of the extremities and other neurological symptoms and signs, and finally did not respond to any
stimuli within a year. All showed characteristic PSD patterns in EEG and rapidly progressing brain atrophy on repeated MRI or CT scan examinations. The patients were then in a vegetative state and died 4 months to 3 years after the initial symptoms. Postmortem examination of the brains was performed in all four cases and the clinical diagnosis was confirmed by neuropathological examination (panencephalopathic type of CJD in Cases 1 and 2, and spongiform encephalopathy in Cases 3 and 4) [4, 9]. The clinical diagnosis of SDAT was based on the criteria by McKhann et al. [8] and on MR! and/or CT scan findings. The CSF ubiquitin levels of the controls ranged from 7.3 to 21.0 ng/ml, 14.3 z 1.1 ng/ml in the mean 7- S.E M. (Fig. 1). The CSF ubiquitin concentrations in the cases of M I D and SDAT were 16.6 z 6.4 ng/ml and 2t.3 ~- 6.1 ng/ml, respectively (Fig. 1L in the CJD cases, the CSF levels of ubiquitin were markedly elevated ranging from 107.6 to 377.0 ng/ml at the early and middle stages of the disease (230.6 ng/ml in Case 1, 107.6 ng/ml in Case 2. 212.5 ng/ml in Case 3 and 377.0 ng, ml in Case 4) (Fig. 1). No, or only a mild, increase m CSF protein was observed in these cases. In Cases 1 and 2. we could examine CSF samples at several different points of the clinical course and the CSF ubiquitin values were found to decrease gradually as the disease progressed (Fig. 2). Since ubiquitin is required for degradation of abnormal proteins in cells [1, 3], the CSF ubiquitin increase in CJD may be due to its overproduction in CNS in response to noxious agentls), such as prion, and its leakage fi'om CNS to CSF. In this regard, of particular
49
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