Acta Neuropathologica

, Volume 86, Issue 4, pp 319–328

Ultrastructural observations on neuronal lipofuscin (age pigment) and dense bodies induced by a proteinase inhibitor, leupeptin, in rat hippocampus

  • A. Nunomura
  • T. Miyagishi
Regular Papers


The ultrastructure of lipofuscin (age pigment) and dense bodies induced by intraventricular administration of leupeptin, a cysteine proteinase inhibitor, were investigated in the neurons of rat hippocampal dentate gyrus. Four-day treatment with leupeptin (0.5 mg/day) rapidly caused a considerable accumulation of intracytoplasmic dense bodies and swelling of neuronal processes. We demonstrated, as inner structures of the pigments, that pentalaminar structure with a thickness of 12–13 nm and finely granular matrix were exactly common to the leupeptin-induced dense bodies and lipofuscin granules. Furthermore, the transitional stages from lysosomes into the dense granules were observed in the neurons of the leupeptin-treated rats. On the other hand, some morphological differences between the leupeptin-induced dense bodies and lipofuscin granules have been shown: (1) distribution in different cell types, (2) intracytoplasmic location, (3) tendencies to associate with vacuoles, and (4) electron density. The present findings suggested that the decline of the lysosomal protein degradation could play a role in lipofuscinogenesis, especially in the genesis of their electron-dense portion, but some other mechanisms might participate in the formation and accumulation of lipofuscin with aging

Key words

Leupeptin Protease inhibitor Lipofuscin Rat hippocampus Aging 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Boellaard JW, Schlote W (1986) Ultrastructural heterogeneity of neuronal lipofuscin in the normal human cerebral cortex. Acta Neuropathol (Berl) 71:285–294Google Scholar
  2. 2.
    Braak H (1971) Über das Neurolipofuscin in der unteren Olive und dem Nucleus dentatus cerebelli im Gehirn des Menschen. Z Zellforsch 121:573–592Google Scholar
  3. 3.
    Brunk U, Ericsson JLE (1972) Electron microscopical studies on rat brain neurons. Localization of acid phosphatase and mode of formation of lipofuscin bodies. J Ultrastruct Res 38:1–15Google Scholar
  4. 4.
    Chio KS, Reiss U, Fletcher B, Tappel AL (1969) Peroxidation of subcellular organelles: formation of lipofuscinlike fluorescent pigments. Science 166:1535–1536Google Scholar
  5. 5.
    Constantinides P, Harkey M, McLaury D (1986) Prevention of lipofuscin development in neurons by anti-oxidants. Virchows Arch [A] 409:583–593Google Scholar
  6. 6.
    Goldspink DF, Lewis SEM (1985) Age-and activity-related changes in three proteinase enzymes of rat skeletal muscle. Biochem J 230:833–836Google Scholar
  7. 7.
    Hasan M, Glees P (1972) Electron microscopical appearance of neuronal lipofuscin using different preparative techniques including freeze-etching. Exp Gerontol 7:345–351Google Scholar
  8. 8.
    Heinsen H (1979) Lipofuscin in the cerebellar cortex of albino rats: an electron microscopic study. Anat Embryol 155:333–345Google Scholar
  9. 9.
    Holtzman E, Novikoff AB (1965) Lysosomes in the rat sciatic nerve following crush. J Cell Biol 27:651–669Google Scholar
  10. 10.
    Ivy GO (1992) Protease inhibitors as a model for NCL disease, with special emphasis on the infantile and adult forms. Am J Med Genet 42:555–560Google Scholar
  11. 11.
    Ivy GO, Gurd JW (1988) A proteinase inhibitor model of lipofuscin formation. In: Zs-Nagy I (ed) Lipofuscin-1987: state of the art. Excerpta Medica, Amsterdam, pp 83–108Google Scholar
  12. 12.
    Ivy GO, Schottler F, Wenzel J, Baudry M, Lynch G (1984) Inhibitors of lysosomal enzymes: accumulation of lipofuscinlike dense bodies in the brain. Science 226:985–987Google Scholar
  13. 13.
    Ivy GO, Kanai S, Ohta M, Smith G, Sato Y, Kobayashi M, Kitani K (1990) Lipofuscin-like substances accumulate rapidly in brain, retina and internal organs with cysteine proteasse inhibition. In: Porta EA (ed) Lipofuscin and ceroid pigments. Plenum Press, New York, pp 31–47Google Scholar
  14. 14.
    Ivy GO, Kanai S, Ohta M, Sato Y, Otsubo K, Kitani K (1991) Leupeptin causes an accumulation of lipofuscin-like substances in liver cells of young rats. Mech Ageing Dev 57:213–231Google Scholar
  15. 15.
    Jolly RD, Barns G, Bube A, Palmer DN (1987) Ovine ceroid-lipofuscinosis: chemical constituents of the lipopigment, their pathogenic significance and similarities to age pigment. In: Totaro EA, Glees P, Pisanti FA (eds) Advances in age pigments research. Pergamon press, Oxford, pp 197–215Google Scholar
  16. 16.
    Kirschke H, Langner J, Reimann S, Wiederanders B, Ansorge S, Bohley P (1980) Lysosomal cysteine proteinases. In: Evered D, Whelan J (eds) Protein degradation in health and disease. Excerpta Medica, Amsterdam, pp 15–35Google Scholar
  17. 17.
    Landfield PW, Braun LD, Pitler TA, Lindsey JD, Lynch G (1981) Hippocampal aging in rats: a morphometric study of multiple variables in semithin sections. Neurobiol Aging 2:265–275Google Scholar
  18. 18.
    Mann DMA, Yates PO (1974) Lipoprotein pigments-their relationship to ageing in the human nervous system. Brain 97:481–488Google Scholar
  19. 19.
    Marzabadi MR, Sohal RS, Brunk UT (1991) Mechanisms of lipofuscinogenesis: effect of the inhibition of lysosomal proteinases and lipases under varying concentrations of ambient oxygen in cultured rat neonatal myocardial cells. APMIS 99:416–426Google Scholar
  20. 20.
    Miquel J, Oro J, Bensch KG, Johnson JE Jr (1977) Lipofuscin: fine-structural and biochemical studies. In: Pryor WA (ed) Free radicals in biology. Academic Press, New York, pp 133–182Google Scholar
  21. 21.
    Miyagishi T, Takahata N, Iizuka R (1967) Electron microscopic studies on the lipo-pigments in the cerebral cortex nerve cells of senile and vitamin Edeficient rats. Acta Neuropathol (Berl) 9:7–17Google Scholar
  22. 22.
    Nandy K (1971) Properties of neuronal lipofuscin pigment in mice. Acta Neuropathol (Berl) 19:25–32Google Scholar
  23. 23.
    Nishioka N, Takahata N, Iizuka R (1968) Histochemical studies on the lipo-pigments in the nerve cells. A comparison with lipofuscin and ceroid pigment. Acta Neuropathol (Berl) 11:174–181Google Scholar
  24. 24.
    Nunomura A (1991) Morphological evaluation of the effects of 7-hydroxy-1-[4-(3-methoxyphenyl)-1-piperazinyl]-acetylamino-2,2,4,6-tetramethylindan (OPC-14117) with anti-oxidant properties on lipofuscin in rat cerebral cortex. Neuropathology 11:89–104 (in Japanese)Google Scholar
  25. 25.
    Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates, 2nd edn. Academic Press, SydneyGoogle Scholar
  26. 26.
    Sarkis GJ, Ashcom JD, Hawdon JW, Jacobson LA (1988) Decline in protease activities with age in the nematode Caenorhabditis elegans. Mech Ageing Dev 45:191–201Google Scholar
  27. 27.
    Schlote W, Boellaard JW (1983) Role of lipopigment during aging of nerve and glial cells in the human central nervous system. In: Cervós-Navarro J, Sarkander HI (eds) Brain aging: neuropathology and neuropharmacology. Raven Press, New York, pp 27–74Google Scholar
  28. 28.
    Sekhon SS, Maxwell DS (1974) Ultrastructural changes in neurons of the spinal anterior horn of ageing mice with particular reference to the accumulation of lipofuscin pigment. J Neurocytol 3:59–72Google Scholar
  29. 29.
    Strehler BL, Mark DD, Mildvan AS, Gee MV (1959) Rate and magnitude of age pigment accumulation in the human myocardium. J Gerontol 14:430–439Google Scholar
  30. 30.
    Takauchi S, Miyoshi K (1989) Degeneration of neuronal processes in rats induced by a protease inhibitor, leupeptin. Acta Neuropathol 78:380–387Google Scholar
  31. 31.
    Thaw HH, Collins VP, Brunk UT (1984) Influence of oxygen tension, pro-oxidants and antioxidants on the formation of lipid peroxidation products (lipofuscin) in individual cultivated human glial cells. Mech Ageing Dev 24:211–223Google Scholar
  32. 32.
    Toyo-oka T, Shimizu T, Masaki T (1978) Inhibition of proteolytic activity of calcium activated neutral protease by leupeptin and antipain. Biochem Biophys Res Commun 82:484–491Google Scholar
  33. 33.
    Wisniewski HM, Wen GY (1988) Lipopigment in the aging brain. Am J Med Genet [Suppl] 5:183–191Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • A. Nunomura
    • 1
  • T. Miyagishi
    • 1
  1. 1.Department of Psychiatry and NeurologyAsahikawa Medical CollegeAsahikawaJapan

Personalised recommendations