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Ultrastructural morphology of cytoplasmic inclusions within neurons of ageing mice
123
Journal of the neurological Sciences
Elsevier Publishing Company, Amsterdam, Printed in The Netherlands
Ultrastructural Morphology of Cytoplasmic Inclusions within Neurons of Ageing Mice H. FRASER, W. S M I T H AND E. W. G R A Y A.R.C. Animal Breeding Research Organisation (H.F.) and Moredun Research Institute, Gilmerton, Edinburgh (Great Britain)
(Received 6 September, 1969)
INTRODUCTION
Eosinophilic inclusions within the cytoplasm of neurons in the thalamus of ageing mice have been described. These are round, oval or disc-shaped bodies ranging in size up to 25 # m in diameter, sometimes having a pale centre or containing within them a basophilic strand or spot. They are confined to the dorsal and ventral thalamic nuclei and are most frequently seen in mice over 1 year of age. They contain protein, and are seen in both sexes of many strains of mice (FRASER 1969). The present report is a result of an investigation of the ultra-structure of these eosinophilic, protein-containing bodies.
MATERIALS AND METHODS
Two 2-year-old female C57BL mice were killed by decapitation and blocks from the thalamus were placed into fixative within 1 min of death. Fixation was in 3 ~ gluteraldehyde followed by post-fixation in 1 ~ osmium tetroxide. Blocks from the thalamic region were embedded in araldite, and 0.5-1 /zm thick sections were stained with toluidine blue and examined with the optical microscope. When the neuronal inclusions were identified the blocks were trimmed down to the relevant area and sections prepared for electron microscopy.
RESULTS
The inclusions which were identified in the thick sections with the optical microscope were sometimes homogeneous, or contained a pale centre, and lay within the cytoplasm of neurons and stained greenish blue with toluidine blue (Fig. 1). Ultrastructurally these bodies consist of arrays of parallel fibrillar crystalline Z neurol. ScL (1970) 11:123-127
Fig. 1. Optical microscopic appearance o f inclusion within cytoplasm of neuron. Toluidine blue: ~.:i 800.
Fig. 2. Electron-microscopic appearance of same inclusion as in Fig. I. :: 15,000.
Fig. 3. Electron-microscopic appearance of inclusion showing fibrillar crystalline material arranged in bundles, x 45,000.
Fig.4. Electron micrograph showing transverse and longitudinal striations, x 105,000.
126
H. FRASER, W. SMITH, E. W. GRAY
material with a well defined substructure, consisting of longitudinal and transverse striations (Fig. 2). The longitudinal striations consist of alternating electron-dense and electron-transparent lines, the distance between the centre of one electron-dense line and the next being 15 nm. The transverse striations are less obvious and appear to result from an alternate narrowing and widening of the longitudinal lines, the periodicity of which is in phase across the thickness of the fibre (Fig. 3 and 4). Where bundles of fibres are separated from one another faint lines can be discerned which frequently appear to be continuous with the longitudinal striations of the individual fibres (Fig. 2).
DISCUSSION
The ultrastructure of these inclusions supports the conclusion that they consist of protein (FRASER 1969). They are distinguishable both from the laminated bodies described within neurons in the cerebellum and lateral geniculate body of the cat (MORALES AND DUNCAN 1966; MORALES et al. 1964) and from the cytoplasmic laminar bodies in the visual cortex of the monkey (KRUGER A~D MAXWELL1969). They can also be distinguished from various crystalline and developmental forms of lipofuscin described in ageing mice (SAMORAJSKIet al. 1965). It appears from their structure that these bodies form from the union of many identical macromolecules. Their similarity between different cells and their homogeneity between and within various host genotypes (FRASER 1969) strongly suggests that they represent an expression of some normal cell activity. Why, then, do they occur in association with ageing? They may represent the accumulation with the passage of time of a normal metabolite; or there may be a reduction, v,ith age, in the homeostatic mechanisms which catalyse their removal. Alternatively they may reflect a reduced cell function with age unaccompanied by a commensurate reduction in the synthesis of the particular macromolecule. It is unlikely that these protein masses represent the morphological equivalent of those abnormal proteins which, it has been suggested, should be biochemically demonstrable in ageing cells (HOLLIDAV 1969), because this hypothesis assumes an accumulation of errors in the specificity of the enzymes responsible for gene translation, resulting in the synthesis of various proteins which are surplus or irrelevant to the functional requirements of the cell and predicts the presence of many different types of protein. Alternatively it might be postulated that these inclusions represent a protein of virus origin caused by an unidentified latent agent, similar to the intranuclear crystalline protein seen in adenovirus infection (MORGAN et al. 1960). If the protein of these inclusions is in all cases identical this would support the concept of an endosymbiotic relationship with a' specific latent virus, but if variation occurs this would suggest that it represents a morphological example of the molecular chaos which occurs with ageing (MEDVEDEV 1966, 1967).
ACKNOWLEDGEMENT
The authors wish to thank Mrs. P. M. Scarborough for the photographs. J. neurol. Sci. (11970) 1 1 : 1 2 3 - 1 2 7
ULTRASTRUCTURALMORPHOLOGY OF CYTOPLASMICINCLUSIONS WITHIN NEURONS
127
SUMMARY Eosinophilic inclusions occur within the cytoplasm of thalamic n e u r o n s of aged mice. T h e ultrastructure of these inclusions as seen in two 2-year-old C57BL mice, is described. Their structure, arrays of parallel fibrillar material with repeating longit u d i n a l a n d transverse striations, confirms a protein consistency, a n d suggests that they form f r o m the u n i o n of m a n y identical macromolecules. The identical n a t u r e o f the bodies between different cells a n d hosts suggests that they are the result of n o r m a l cell activity, a n d represent the a c c u m u l a t i o n with time of a n o r m a l metabolite or a r e t a r d a t i o n of homeostatic m e c h a n i s m s which catalyse their removal.
REFERENCES
FRASER,H. (1969) Eosinophilic bodies in some neurones in the thalamus of ageing mice, J. Path., 98 : 201-204. HOLLIDAY, R. (1969) Errors in protein synthesis and clonal senescence in fungi, Nature (Lond.), 221 : 1224-1228. KRUGER,L. AND D. S. MAXWELL(1969) Cytoplasmic laminar bodies in the striate cortex, J. Ultrastruet. Res., 26: 387-390. MEDVEDEV,Z. A. (1966) In: Protein Biosynthesis and Problems of Heredity, Development, and Ageing, Oliver and Boyd, Edinburgh, London, p. 498. MEDVEDEV,Z. A. (1967) Molecular aspects of ageing. In: H. W. WOOLHOUSE(Ed.), Aspects of the Biology of Ageing (21st Symposium of the Society of Experimental Biology), Cambridge University Press, Cambridge, Mass., pp. 1-28. MORALES,R. AND D. DUNCAN(1966) Multilaminated bodies and other unusual configurations of endoplasmic reticulum in the cerebellum of the cat. An electron microscopic study, J. UItrastruct. Res., 15: 480-489. MORALES,R., D. DUNCANAND R. REHMET(1964) A distinctive laminated cytoplasmic body in the lateral geniculate body neurons of the cat, J. Ultrastruet. Res., 10:116-123. MORGAN, C., G. C. GODMAN,P. M. BRIETENFELDAND H. M. ROSE (1960) A correlative study by electron and light microscopy of the development of type 5 adenovirus, Part 1 (Electron microscopy), J. exp. Med., 112: 373-382. SAMORAJSKI,T,, J. M. ORDY AND J. R. KEEFE(1965) The fine structure of lipofuscin age pigment in the nervous system of aged mice, J. Cell Biol., 26: 779-795.
J. neurol. Sci. (1970) 11:123-127
Journal of the neurological Sciences
Elsevier Publishing Company, Amsterdam, Printed in The Netherlands
Ultrastructural Morphology of Cytoplasmic Inclusions within Neurons of Ageing Mice H. FRASER, W. S M I T H AND E. W. G R A Y A.R.C. Animal Breeding Research Organisation (H.F.) and Moredun Research Institute, Gilmerton, Edinburgh (Great Britain)
(Received 6 September, 1969)
INTRODUCTION
Eosinophilic inclusions within the cytoplasm of neurons in the thalamus of ageing mice have been described. These are round, oval or disc-shaped bodies ranging in size up to 25 # m in diameter, sometimes having a pale centre or containing within them a basophilic strand or spot. They are confined to the dorsal and ventral thalamic nuclei and are most frequently seen in mice over 1 year of age. They contain protein, and are seen in both sexes of many strains of mice (FRASER 1969). The present report is a result of an investigation of the ultra-structure of these eosinophilic, protein-containing bodies.
MATERIALS AND METHODS
Two 2-year-old female C57BL mice were killed by decapitation and blocks from the thalamus were placed into fixative within 1 min of death. Fixation was in 3 ~ gluteraldehyde followed by post-fixation in 1 ~ osmium tetroxide. Blocks from the thalamic region were embedded in araldite, and 0.5-1 /zm thick sections were stained with toluidine blue and examined with the optical microscope. When the neuronal inclusions were identified the blocks were trimmed down to the relevant area and sections prepared for electron microscopy.
RESULTS
The inclusions which were identified in the thick sections with the optical microscope were sometimes homogeneous, or contained a pale centre, and lay within the cytoplasm of neurons and stained greenish blue with toluidine blue (Fig. 1). Ultrastructurally these bodies consist of arrays of parallel fibrillar crystalline Z neurol. ScL (1970) 11:123-127
Fig. 1. Optical microscopic appearance o f inclusion within cytoplasm of neuron. Toluidine blue: ~.:i 800.
Fig. 2. Electron-microscopic appearance of same inclusion as in Fig. I. :: 15,000.
Fig. 3. Electron-microscopic appearance of inclusion showing fibrillar crystalline material arranged in bundles, x 45,000.
Fig.4. Electron micrograph showing transverse and longitudinal striations, x 105,000.
126
H. FRASER, W. SMITH, E. W. GRAY
material with a well defined substructure, consisting of longitudinal and transverse striations (Fig. 2). The longitudinal striations consist of alternating electron-dense and electron-transparent lines, the distance between the centre of one electron-dense line and the next being 15 nm. The transverse striations are less obvious and appear to result from an alternate narrowing and widening of the longitudinal lines, the periodicity of which is in phase across the thickness of the fibre (Fig. 3 and 4). Where bundles of fibres are separated from one another faint lines can be discerned which frequently appear to be continuous with the longitudinal striations of the individual fibres (Fig. 2).
DISCUSSION
The ultrastructure of these inclusions supports the conclusion that they consist of protein (FRASER 1969). They are distinguishable both from the laminated bodies described within neurons in the cerebellum and lateral geniculate body of the cat (MORALES AND DUNCAN 1966; MORALES et al. 1964) and from the cytoplasmic laminar bodies in the visual cortex of the monkey (KRUGER A~D MAXWELL1969). They can also be distinguished from various crystalline and developmental forms of lipofuscin described in ageing mice (SAMORAJSKIet al. 1965). It appears from their structure that these bodies form from the union of many identical macromolecules. Their similarity between different cells and their homogeneity between and within various host genotypes (FRASER 1969) strongly suggests that they represent an expression of some normal cell activity. Why, then, do they occur in association with ageing? They may represent the accumulation with the passage of time of a normal metabolite; or there may be a reduction, v,ith age, in the homeostatic mechanisms which catalyse their removal. Alternatively they may reflect a reduced cell function with age unaccompanied by a commensurate reduction in the synthesis of the particular macromolecule. It is unlikely that these protein masses represent the morphological equivalent of those abnormal proteins which, it has been suggested, should be biochemically demonstrable in ageing cells (HOLLIDAV 1969), because this hypothesis assumes an accumulation of errors in the specificity of the enzymes responsible for gene translation, resulting in the synthesis of various proteins which are surplus or irrelevant to the functional requirements of the cell and predicts the presence of many different types of protein. Alternatively it might be postulated that these inclusions represent a protein of virus origin caused by an unidentified latent agent, similar to the intranuclear crystalline protein seen in adenovirus infection (MORGAN et al. 1960). If the protein of these inclusions is in all cases identical this would support the concept of an endosymbiotic relationship with a' specific latent virus, but if variation occurs this would suggest that it represents a morphological example of the molecular chaos which occurs with ageing (MEDVEDEV 1966, 1967).
ACKNOWLEDGEMENT
The authors wish to thank Mrs. P. M. Scarborough for the photographs. J. neurol. Sci. (11970) 1 1 : 1 2 3 - 1 2 7
ULTRASTRUCTURALMORPHOLOGY OF CYTOPLASMICINCLUSIONS WITHIN NEURONS
127
SUMMARY Eosinophilic inclusions occur within the cytoplasm of thalamic n e u r o n s of aged mice. T h e ultrastructure of these inclusions as seen in two 2-year-old C57BL mice, is described. Their structure, arrays of parallel fibrillar material with repeating longit u d i n a l a n d transverse striations, confirms a protein consistency, a n d suggests that they form f r o m the u n i o n of m a n y identical macromolecules. The identical n a t u r e o f the bodies between different cells a n d hosts suggests that they are the result of n o r m a l cell activity, a n d represent the a c c u m u l a t i o n with time of a n o r m a l metabolite or a r e t a r d a t i o n of homeostatic m e c h a n i s m s which catalyse their removal.
REFERENCES
FRASER,H. (1969) Eosinophilic bodies in some neurones in the thalamus of ageing mice, J. Path., 98 : 201-204. HOLLIDAY, R. (1969) Errors in protein synthesis and clonal senescence in fungi, Nature (Lond.), 221 : 1224-1228. KRUGER,L. AND D. S. MAXWELL(1969) Cytoplasmic laminar bodies in the striate cortex, J. Ultrastruet. Res., 26: 387-390. MEDVEDEV,Z. A. (1966) In: Protein Biosynthesis and Problems of Heredity, Development, and Ageing, Oliver and Boyd, Edinburgh, London, p. 498. MEDVEDEV,Z. A. (1967) Molecular aspects of ageing. In: H. W. WOOLHOUSE(Ed.), Aspects of the Biology of Ageing (21st Symposium of the Society of Experimental Biology), Cambridge University Press, Cambridge, Mass., pp. 1-28. MORALES,R. AND D. DUNCAN(1966) Multilaminated bodies and other unusual configurations of endoplasmic reticulum in the cerebellum of the cat. An electron microscopic study, J. UItrastruct. Res., 15: 480-489. MORALES,R., D. DUNCANAND R. REHMET(1964) A distinctive laminated cytoplasmic body in the lateral geniculate body neurons of the cat, J. Ultrastruet. Res., 10:116-123. MORGAN, C., G. C. GODMAN,P. M. BRIETENFELDAND H. M. ROSE (1960) A correlative study by electron and light microscopy of the development of type 5 adenovirus, Part 1 (Electron microscopy), J. exp. Med., 112: 373-382. SAMORAJSKI,T,, J. M. ORDY AND J. R. KEEFE(1965) The fine structure of lipofuscin age pigment in the nervous system of aged mice, J. Cell Biol., 26: 779-795.
J. neurol. Sci. (1970) 11:123-127