319
Brain Research, 551 (1991) 319-321 ~) 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A D ONIS 000689939124699K
BRES 24699
Buffy coat from families of Alzheimer's disease patients produces intracytoplasmic neurofilament accumulation in hamster brain Masatoshi Takeda, Tsuyoshi Nishimura, Takashi Kudo, Satoshi Tanimukai and Kunitoshi Tada Department of Neuropsychiatry, Osaka University Medical School, Fukushima, Osaka (Japan)
(Accepted 12 March 1991) Key words: Alzheimer's disease; Neurofilament; Transmission; Buffy coat
Buffy coat of three members from a family with Alzheimer's disease was inoculated into hamster brains. Eighteen months after the inoculation, all experimental animals were sacrificed for the neuropathological study. Hematoxylin-eosin staining showed no gross vacuolar degeneration, or neuronal loss in the cortex. The spongiform degeneration was minimum. Immunostaining with antibodies against neurofilament 200 kDa subunit protein revealed massive immuno-positive intracytoplasmic inclusion bodies within neurons of the brainstem nuclei. By electron microscopy, the intracytoplasmic inclusion body was shown to be composed of proliferated 10 nm neurofilaments. The intra-cytoplasmic neurofilament proliferation was observed with the hamsters inoculated with the buffy coat from Alzheimer's disease patients as well as an apparently normal member of the family.
Alzheimer's disease, a major cause of dementia in presenile and senile population, still remains a disease of unknown etiology. Transmission studies of Alzheimer's disease have been tried by several investigators, but none has succeeded in transmitting it to convenient laboratory animals from the diseased brain tissue 4'9. In 1988 Manuelidis et al. reported the results of the transmission study from buffy coat obtained from blood of Alzheimer patients and healthy relatives. Out of 11 relatives of Alzheimer patients, 5 individuals produced histologically documented spongiform encephalopathy in recipient hamsters 1°, though their results have not been confirmed by others. Since the results of his research are so important, we designed an inoculation study of the buffy coat obtained from members of a family with Alzheimer's disease. Three members from a family with Alzheimer's disease and two age-matched apparently healthy control subjects participated in the study. No. 1 and No. 2 are brothers with Alzheimer's disease diagnosed by NINCSA D R D A criteria 8. Before the project was completed, both of them were dead and the neuropathological examination of the autopsied brain of No. 1 confirmed the diagnosis of Alzheimer's disease. No. 3 is the daughter of No. 1, who is apparently normal by neuropsychiatric evaluation. No. 4 and No. 5 are genetically
unrelated healthy control subjects. Twenty ml of blood was drawn from each volunteer into a sterile heparinized tube, centrifuged at 1000 rpm for 15 min. Under the strict sterile conditions, the bully coat was aspirated by a capillary, mixed with the same volume of saline, and homogenized. The homogenate was injected i.c.v, into each of four 6-week-old Golden hamsters per human sample 11. Four hamsters were kept in a numbered cage and were observed for any unusual signs by technicians. Eighteen months after the inoculation, all hamsters were sacrificed for the neuropathological study. The serial coronal sections throughout the brain and the cervical spinal cord were studied by hematoxylin-eosin and the immunostaining. Monoclonai antibodies used in this study were antibodies against the neurofilament 200 kDa subunit protein obtained from two different sources 3 (Amersham and Sigma). The brain tissue was immunostained by the ABC method according to the instructions of Vectastain Elite ABC kit (Vector Labs, Burlingame, CA) 5. By 18 months after the inoculation, 5 out of 20 hamsters were dead. In total 18 hamsters were studied, including the three which were sacrificed on 83,277, and 289 days after the inoculation. The buffy coat obtained from members of the Alzheimer family (Nos. 1-3) all produced abnormal accumulation of neurofilaments in
Correspondence: M. Takeda, Department of Neuropsychiatry, Osaka University Medical School, Fukushima, Osaka 553, Japan. Fax: (81) (6) 451-4072.
320
Fig. l. Immunostaining of vestibular nucleus of hamster inoculated with buffy coat from No. 1 with anti-neurofilament 200 kUa protein antibody (Amersham), ×75.
neuronal p e r i k a r y a of the brainstem nuclei, most prominent in the vestibular nuclei (Fig. 1). W h e n the immunohistological study revealed the immunopositive neurons in the brainstem nuclei by immunostaining with an antibody against neurofilament 200 k D a subunit protein ( A m e r s h a m ) , the hamster was designated as positive. Six out of 12 hamsters inoculated with the buffy coat from the A l z h e i m e r family m e m b e r s were positive, while all of the hamsters inoculated with the buffy coat from the control subjects were negative (Table I). Intracytoplasmic accumulation of neurofilament was confirmed by immunostaining with a different monoclonal antibody against neurofilament 200 k D a subunit protein (Sigma) (Fig. 2). The immunopositive neurons were observed in coronal sections lower than the level of the nucleus trigemini in the m e t e n c e p h a l o n , including the nuclei nervi VIII, X, and XII. In sections higher than the metencephalon, no immunopositive neurons were observed including cerebral cortex, hippocampus, and cerebellum. The electron microscopic observation revealed massive accumulation of 10 nm neurofilament-like fibers in neuronal cytoplasm (Fig. 3). The accumulated neurofilament-like fibers were
Fig. 2. Higher magnification of immunopositivc neuron with antineurofilament 200 kDa protein antibody (Sigma). ×700. indistinguishable from the normal neurofilanaent fibers. The most m a r k e d neuropathological finding with the positive h a m s t e r brain was the intracytoplasmic neurofilament accumulation in neuronal p e r i k a r y a of the lower brainstem nuclei. Changes indicating spongiform encephalopathy or granulovacuolar d e g e n e r a t i o n were minimum. The a b n o r m a l intracytoplasmic neurofilament accumulation in neurons of the lower brainstem nuclei was observed bilaterally, though the n u m b e r of immunopositive neurons were more in the ipsilateral side to the inoculation. Three hamsters inoculated with the buffy coat from A l z h e i m e r patients, which had to be sacrificed on 83,277, and 389 days due to severe wound caused by cage mates, were all negative. Since the neurofilament accumulation was observed bilaterally and the location of the accumulated neurofilaments was far from the inoculation site, it is unlikely that the neurofilament accumulation is due to nonspecific response of the brain tissue which was triggered by traumatic insult. As for the question of disease specificity, it is strongly indicated that the intra-cyto
TABLE I Numbers of positive animals by buffy coat inoculation
+, Positive; -, negative; f.d., found dead. Volunteer No.
Sex
Age
Results
1 2 3 4 5
M M F M F
55 52 24 54 53
1+ / / 3 f.d. 2+ / 23+ / / 1 f.d. / 3-/ If.d. / 4-
(Alzheimer) (Alzheimer) (daughter of l) (healthy control) (healthy control)
Fig. 3. Electron microscopy of accumulated neurofilaments by inoculation of buffy coat from Alzheimer's disease patient No. 2.
321 plasmic inclusion body composed of neurofilaments is produced by a factor present in the buff3' coat of Alzheimer patients and families of the patients, because the inoculation of the buffy coats from the control subjects did not produce any positive results. Since only one family was used in this study, it is hard to determine whether the pathological changes observed in this study were related to peculiarities of the genetic and environmental background of a single family or to something c o m m o n to Alzheimer's disease. It is to be noticed that the same positive result was obtained by the buffy coat obtained from the apparently healthy daughter of the patient. It is possible that factors in family environment or genetic traits are shared by the patients and the daughter. Though Manuelidis et al. reported the production of spongiform encephalopathy in the hamster brains after inoculation of buffy coats of Alzheimer patients 1°, the present study shows quite different neuropathologicai findings. The major finding is massive neurofilament accumulation in neuronal perikarya of the lower brainstem nuclei. The intracytoplasmic neurofilament accumulation has been well documented in the rabbit brain intoxicated with aluminum 2'6'7A2. In fact, aluminum-induced experimental neurofibrillary degeneration has been studied
intensively as an animal model of Alzheimer pathology 1. Comparing with our experience with aluminum-intoxicated rabbit brains 13'14 and those in the literature 7'12, the hamster brain inoculated with Alzheimer buffy coat shows the more massive intracytoplasmic neurofilament accumulation. The distribution pattern of the accumulated neurofilament is, however, similar between these two experimental animals. The present results demonstrate that the buffy coat from Alzheimer patients as well as family members of the patients can produce abnormal neurofilament accumulation in neuronal perikarya of the lower brainstem nuclei. It is still an open question whether this abnormal neurofilament accumulation is due to any exogenous transmissible agents shared by the family environment or any genetically transmitted factors. Even though the experiment was with only one family of Alzheimer's disease, it is an important finding that the buffy coat obtained from Alzheimer patients as well as an apparently normal family m e m b e r causes definitely pathological response in the hamster brain.
1 Crapper, D.R., Krishnan, S.S. and Quittkat, S., Aluminum, neurofibrillary degeneration and Alzheimer disease, Brain, 99 (1976) 67-79. 2 De Boni, U., Otvas, A., Scott, J.W. and Crapper, D.R., Neurofibrillary degeneration induced by systemic aluminum, Acta Neuropathol., 35 (1976) 285-294. 3 Debus, E., Weber, K. and Osborn, M., Monoclonal antibodies specific to GFA protein and for each of the neurofilament polypeptides, Differentiation, 25 (1983) 193-203. 4 Goudsmit, J., Morrow, C.H., Asher, D.M., Yanagihara, R.T., Masters, C.L., Gibbs, C.J. and Gajdusek, D.C., Evidence for and against the transmissibility of Alzheimer's disease, Neurology, 30 (1980) 945-950. 5 Hsu, S., Raine, L. and Fanger, H., Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 6 Klatzo, I., Wisniewski, H. and Streicher, E., Experimental production of neurofibrillary degeneration. I. Light microscopic observations, J. Neuropathol. Exp. Neurol., 24 (1965) 187-199. 7 Kowall, N.W., Pendlebury, W.W., Kessler, J.B., Perl, D.P. and Beal, M.E, Aluminium-induced neurofibrillary degeneration affects a subset of neurons in rabbit cerebral cortex, basal forebrain, and upper brainstem, Neuroscience, 29 (1989) 329337.
8 McKhann, G., Drachman, D., Folstein, M.E, Katzman, R., Price, D. and Stadlan, E., Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA work group under the auspices of the Department of Health and Human Services Task Force on Alzheimer's disease, Neurology, 14 (1984) 284-293. 9 Manuelidis, E.E., Creutzfeldt-Jakob disease, J. Neuropathol. Exp. Neurol., 44 (1985) 1-17. 10 Manuelidis, E.E., De Figueiredo, J.M., Kim, J.H., Fritch, W.W. and Manuelidis, L., Transmission studies from blood of Alzheimer disease patients and healthy relatives, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 4898-4901. 11 Manuelidis, E.E., Gorgacz, E.J. and Manuelidis, L., Viremia in experimental Creutzfeldt-Jakob disease, Science, 200 (1978) 1069-1071. 12 Selkoe, D.J., Liem, R.K.H., Yen, S. and Shelanski, M.L., Biochemical and immunological characterization of neurofilaments in experimental neurofibrillary degeneration by aluminum, Brain Research, 163 (1979) 235-252. 13 Suzuki, H., Takeda, M., Nakamura, Y., Tada, K., Hariguchi, S. and Nishimura, T., Activities of enzymes in rabbit brain with experimental neurofibrillary changes, Neurosci. Len., 89 (1988) 234-239. 14 Takeda, M., Experimental study on the mechanism of neurofibrillary change formation, Osaka Univ. Med. J., 34 (1984) 145-161.
Part of this study is supported by grants from the Ministry of Education of Japan, the Ministry of Welfare of Japan, and Sandoz gerontological research foundation.
Brain Research, 551 (1991) 319-321 ~) 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A D ONIS 000689939124699K
BRES 24699
Buffy coat from families of Alzheimer's disease patients produces intracytoplasmic neurofilament accumulation in hamster brain Masatoshi Takeda, Tsuyoshi Nishimura, Takashi Kudo, Satoshi Tanimukai and Kunitoshi Tada Department of Neuropsychiatry, Osaka University Medical School, Fukushima, Osaka (Japan)
(Accepted 12 March 1991) Key words: Alzheimer's disease; Neurofilament; Transmission; Buffy coat
Buffy coat of three members from a family with Alzheimer's disease was inoculated into hamster brains. Eighteen months after the inoculation, all experimental animals were sacrificed for the neuropathological study. Hematoxylin-eosin staining showed no gross vacuolar degeneration, or neuronal loss in the cortex. The spongiform degeneration was minimum. Immunostaining with antibodies against neurofilament 200 kDa subunit protein revealed massive immuno-positive intracytoplasmic inclusion bodies within neurons of the brainstem nuclei. By electron microscopy, the intracytoplasmic inclusion body was shown to be composed of proliferated 10 nm neurofilaments. The intra-cytoplasmic neurofilament proliferation was observed with the hamsters inoculated with the buffy coat from Alzheimer's disease patients as well as an apparently normal member of the family.
Alzheimer's disease, a major cause of dementia in presenile and senile population, still remains a disease of unknown etiology. Transmission studies of Alzheimer's disease have been tried by several investigators, but none has succeeded in transmitting it to convenient laboratory animals from the diseased brain tissue 4'9. In 1988 Manuelidis et al. reported the results of the transmission study from buffy coat obtained from blood of Alzheimer patients and healthy relatives. Out of 11 relatives of Alzheimer patients, 5 individuals produced histologically documented spongiform encephalopathy in recipient hamsters 1°, though their results have not been confirmed by others. Since the results of his research are so important, we designed an inoculation study of the buffy coat obtained from members of a family with Alzheimer's disease. Three members from a family with Alzheimer's disease and two age-matched apparently healthy control subjects participated in the study. No. 1 and No. 2 are brothers with Alzheimer's disease diagnosed by NINCSA D R D A criteria 8. Before the project was completed, both of them were dead and the neuropathological examination of the autopsied brain of No. 1 confirmed the diagnosis of Alzheimer's disease. No. 3 is the daughter of No. 1, who is apparently normal by neuropsychiatric evaluation. No. 4 and No. 5 are genetically
unrelated healthy control subjects. Twenty ml of blood was drawn from each volunteer into a sterile heparinized tube, centrifuged at 1000 rpm for 15 min. Under the strict sterile conditions, the bully coat was aspirated by a capillary, mixed with the same volume of saline, and homogenized. The homogenate was injected i.c.v, into each of four 6-week-old Golden hamsters per human sample 11. Four hamsters were kept in a numbered cage and were observed for any unusual signs by technicians. Eighteen months after the inoculation, all hamsters were sacrificed for the neuropathological study. The serial coronal sections throughout the brain and the cervical spinal cord were studied by hematoxylin-eosin and the immunostaining. Monoclonai antibodies used in this study were antibodies against the neurofilament 200 kDa subunit protein obtained from two different sources 3 (Amersham and Sigma). The brain tissue was immunostained by the ABC method according to the instructions of Vectastain Elite ABC kit (Vector Labs, Burlingame, CA) 5. By 18 months after the inoculation, 5 out of 20 hamsters were dead. In total 18 hamsters were studied, including the three which were sacrificed on 83,277, and 289 days after the inoculation. The buffy coat obtained from members of the Alzheimer family (Nos. 1-3) all produced abnormal accumulation of neurofilaments in
Correspondence: M. Takeda, Department of Neuropsychiatry, Osaka University Medical School, Fukushima, Osaka 553, Japan. Fax: (81) (6) 451-4072.
320
Fig. l. Immunostaining of vestibular nucleus of hamster inoculated with buffy coat from No. 1 with anti-neurofilament 200 kUa protein antibody (Amersham), ×75.
neuronal p e r i k a r y a of the brainstem nuclei, most prominent in the vestibular nuclei (Fig. 1). W h e n the immunohistological study revealed the immunopositive neurons in the brainstem nuclei by immunostaining with an antibody against neurofilament 200 k D a subunit protein ( A m e r s h a m ) , the hamster was designated as positive. Six out of 12 hamsters inoculated with the buffy coat from the A l z h e i m e r family m e m b e r s were positive, while all of the hamsters inoculated with the buffy coat from the control subjects were negative (Table I). Intracytoplasmic accumulation of neurofilament was confirmed by immunostaining with a different monoclonal antibody against neurofilament 200 k D a subunit protein (Sigma) (Fig. 2). The immunopositive neurons were observed in coronal sections lower than the level of the nucleus trigemini in the m e t e n c e p h a l o n , including the nuclei nervi VIII, X, and XII. In sections higher than the metencephalon, no immunopositive neurons were observed including cerebral cortex, hippocampus, and cerebellum. The electron microscopic observation revealed massive accumulation of 10 nm neurofilament-like fibers in neuronal cytoplasm (Fig. 3). The accumulated neurofilament-like fibers were
Fig. 2. Higher magnification of immunopositivc neuron with antineurofilament 200 kDa protein antibody (Sigma). ×700. indistinguishable from the normal neurofilanaent fibers. The most m a r k e d neuropathological finding with the positive h a m s t e r brain was the intracytoplasmic neurofilament accumulation in neuronal p e r i k a r y a of the lower brainstem nuclei. Changes indicating spongiform encephalopathy or granulovacuolar d e g e n e r a t i o n were minimum. The a b n o r m a l intracytoplasmic neurofilament accumulation in neurons of the lower brainstem nuclei was observed bilaterally, though the n u m b e r of immunopositive neurons were more in the ipsilateral side to the inoculation. Three hamsters inoculated with the buffy coat from A l z h e i m e r patients, which had to be sacrificed on 83,277, and 389 days due to severe wound caused by cage mates, were all negative. Since the neurofilament accumulation was observed bilaterally and the location of the accumulated neurofilaments was far from the inoculation site, it is unlikely that the neurofilament accumulation is due to nonspecific response of the brain tissue which was triggered by traumatic insult. As for the question of disease specificity, it is strongly indicated that the intra-cyto
TABLE I Numbers of positive animals by buffy coat inoculation
+, Positive; -, negative; f.d., found dead. Volunteer No.
Sex
Age
Results
1 2 3 4 5
M M F M F
55 52 24 54 53
1+ / / 3 f.d. 2+ / 23+ / / 1 f.d. / 3-/ If.d. / 4-
(Alzheimer) (Alzheimer) (daughter of l) (healthy control) (healthy control)
Fig. 3. Electron microscopy of accumulated neurofilaments by inoculation of buffy coat from Alzheimer's disease patient No. 2.
321 plasmic inclusion body composed of neurofilaments is produced by a factor present in the buff3' coat of Alzheimer patients and families of the patients, because the inoculation of the buffy coats from the control subjects did not produce any positive results. Since only one family was used in this study, it is hard to determine whether the pathological changes observed in this study were related to peculiarities of the genetic and environmental background of a single family or to something c o m m o n to Alzheimer's disease. It is to be noticed that the same positive result was obtained by the buffy coat obtained from the apparently healthy daughter of the patient. It is possible that factors in family environment or genetic traits are shared by the patients and the daughter. Though Manuelidis et al. reported the production of spongiform encephalopathy in the hamster brains after inoculation of buffy coats of Alzheimer patients 1°, the present study shows quite different neuropathologicai findings. The major finding is massive neurofilament accumulation in neuronal perikarya of the lower brainstem nuclei. The intracytoplasmic neurofilament accumulation has been well documented in the rabbit brain intoxicated with aluminum 2'6'7A2. In fact, aluminum-induced experimental neurofibrillary degeneration has been studied
intensively as an animal model of Alzheimer pathology 1. Comparing with our experience with aluminum-intoxicated rabbit brains 13'14 and those in the literature 7'12, the hamster brain inoculated with Alzheimer buffy coat shows the more massive intracytoplasmic neurofilament accumulation. The distribution pattern of the accumulated neurofilament is, however, similar between these two experimental animals. The present results demonstrate that the buffy coat from Alzheimer patients as well as family members of the patients can produce abnormal neurofilament accumulation in neuronal perikarya of the lower brainstem nuclei. It is still an open question whether this abnormal neurofilament accumulation is due to any exogenous transmissible agents shared by the family environment or any genetically transmitted factors. Even though the experiment was with only one family of Alzheimer's disease, it is an important finding that the buffy coat obtained from Alzheimer patients as well as an apparently normal family m e m b e r causes definitely pathological response in the hamster brain.
1 Crapper, D.R., Krishnan, S.S. and Quittkat, S., Aluminum, neurofibrillary degeneration and Alzheimer disease, Brain, 99 (1976) 67-79. 2 De Boni, U., Otvas, A., Scott, J.W. and Crapper, D.R., Neurofibrillary degeneration induced by systemic aluminum, Acta Neuropathol., 35 (1976) 285-294. 3 Debus, E., Weber, K. and Osborn, M., Monoclonal antibodies specific to GFA protein and for each of the neurofilament polypeptides, Differentiation, 25 (1983) 193-203. 4 Goudsmit, J., Morrow, C.H., Asher, D.M., Yanagihara, R.T., Masters, C.L., Gibbs, C.J. and Gajdusek, D.C., Evidence for and against the transmissibility of Alzheimer's disease, Neurology, 30 (1980) 945-950. 5 Hsu, S., Raine, L. and Fanger, H., Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 6 Klatzo, I., Wisniewski, H. and Streicher, E., Experimental production of neurofibrillary degeneration. I. Light microscopic observations, J. Neuropathol. Exp. Neurol., 24 (1965) 187-199. 7 Kowall, N.W., Pendlebury, W.W., Kessler, J.B., Perl, D.P. and Beal, M.E, Aluminium-induced neurofibrillary degeneration affects a subset of neurons in rabbit cerebral cortex, basal forebrain, and upper brainstem, Neuroscience, 29 (1989) 329337.
8 McKhann, G., Drachman, D., Folstein, M.E, Katzman, R., Price, D. and Stadlan, E., Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA work group under the auspices of the Department of Health and Human Services Task Force on Alzheimer's disease, Neurology, 14 (1984) 284-293. 9 Manuelidis, E.E., Creutzfeldt-Jakob disease, J. Neuropathol. Exp. Neurol., 44 (1985) 1-17. 10 Manuelidis, E.E., De Figueiredo, J.M., Kim, J.H., Fritch, W.W. and Manuelidis, L., Transmission studies from blood of Alzheimer disease patients and healthy relatives, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 4898-4901. 11 Manuelidis, E.E., Gorgacz, E.J. and Manuelidis, L., Viremia in experimental Creutzfeldt-Jakob disease, Science, 200 (1978) 1069-1071. 12 Selkoe, D.J., Liem, R.K.H., Yen, S. and Shelanski, M.L., Biochemical and immunological characterization of neurofilaments in experimental neurofibrillary degeneration by aluminum, Brain Research, 163 (1979) 235-252. 13 Suzuki, H., Takeda, M., Nakamura, Y., Tada, K., Hariguchi, S. and Nishimura, T., Activities of enzymes in rabbit brain with experimental neurofibrillary changes, Neurosci. Len., 89 (1988) 234-239. 14 Takeda, M., Experimental study on the mechanism of neurofibrillary change formation, Osaka Univ. Med. J., 34 (1984) 145-161.
Part of this study is supported by grants from the Ministry of Education of Japan, the Ministry of Welfare of Japan, and Sandoz gerontological research foundation.
