156
low molecular weight heparin in patients undergoing total hip replacement. Thromb Haemostas 1988; 60: 407-11. 50. Samama M, Bernard P, Bonnardot JP, Combe-Tamzali S, Lanson Y, Tissot E. Low molecular weight heparin with unfractionated heparin in prevention of postoperative thrombosis. Br J Surg 1988; 75: 128-31. 51. Sasahara AA, Koppenhagen K, Häring R, Welzel D, Wolf H. Low molecular weight heparin plus dihydroergotamine for prophylaxis of postoperative deep vein thrombosis. Br J Surg 1986; 73: 697-700. 52. Verardi S, Casciani CU, Nicora E, et al. A multicentre study on LMW-heparin effectiveness in preventing postsurgical thrombosis. Int Angio 1988; 7 (suppl 3): 19-24. 53. Verardi S, Cortese F, Baroni B, Boffo V, Casciani CU, Palazinni E. Deep vein thrombosis prevention in surgical patients: effectiveness and safety
REVIEW
of
a new
low molecular
weight heparin. Curr Ther Res 1989;
46:
366-72.
Welzel D, Stringer MD, Hedges AR, et al. Fixed combinations of low molecular weight or unfractionated heparin plus dihydroergotamine in the prevention of postoperative deep vein thrombosis. Thromb Haemostas 1989; 62: 523 (abstr). 55. Cruickshank MK, Levine MN, Hirsh J, et al. An elevation of impedance plethysmography and 125-I-fibrinogen legscanning in patients following hip surgery. Thromb Haemostas 1989; 62: 830-36. 56. Laupacis A, Sackett DL, Roberts RS. An assessment of clinically useful measures of the consequences of treatment. N Engl J Med 1988; 318:
54.
1728-33. 57.
Light RS, Pillemer DB. Summing up: the science of reviewing research. Cambridge, Mass: Harvard University Press, 1984: 66-65.
ARTICLE processing in lysosomes: the new therapeutic target in neurodegenerative disease Protein
recognised feature of neurons is their large complement of lysosomes. Studies of the accumulation of the abnormal isoform of the prion protein (PrPSC) in the prion encephalopathies and the formation of &bgr;/A4 protein from its precursor in Alzheimer’s disease suggest that generation of these key proteins takes place in lysosome-related organelles. The release of hydrolytic enzymes from lysosomes may be a primary cause of neuronal damage. Although molecular genetic approaches have identified protein mutations central to the main cell disease, neurodegenerative biological observations are now beginning to unravel the intracellular pathways involved in the molecular pathogenesis of neurodegeneration: as a result, it is now appropriate to consider therapeutic manipulation of the lysosomal system as an approach to treatment. A little
Introduction Neurons do not replicate in adult life, so they need an efficient way of turning over proteins and dealing with any abnormal proteins. To this end they possess a very well-developed lysosome system. Evidence is accumulating to suggest that abortive attempts to degrade proteins within this system lie at the centre of the pathogenesis of some of the major neurodegenerative diseases of man. These include Alzheimer disease and the prion encephalopathies such as Creutzfeldt-Jakob disease where abnormal amyloid (&bgr;/A4) and prion (PrPSC)proteins, respectively, are deposited in and around neurons. This in turn opens up the possibility of new therapeutic strategies, aimed at altering lysosomal
protein processing.
Lysosome system the most familiar part of the large system Lysosomes of acid-containing vesicles that enable cells to digest unwanted material. They are characterised by specific hydro lases (eg, &bgr;-glucuronidase) which are most active at are
low pH. Other components of this acidic vesicle system include endosomes (vesicles formed after membrane intemalisation during receptor-mediated endocytosis), multivesicular and tubulovesicular bodies (which may form by the surface invagination of endosomes), autophagic vacuoles (formed within cells to isolate unwanted organelles), and nascent hydrolase-containing vesicles derived from the protein-packaging Golgi apparatus.l Recent evidence suggests that the lysosome system interacts closely with cell stress proteins. Cell stress proteins-also known as heat-shock proteins (HSP) after one form of cell stress used in early experiments-are highly conserved and have roles in normal cell activity as well as in the protective response to cell damage. They include ubiquitin, a central co-factor in protein degradation, and HSP 70,2 which acts as a molecular "chaperone", facilitating the folding and transport of proteins across different compartments within the cell.3 Initially thought of as cytosolic proteins, both are also found within lysosome related organelles. Immunogold electronmicroscopy has shown that normal lysosomes contain both free ubiquitin’ and ubiquitin-protein conjugates5-7 and that these conjugates accumulate excessively in lysosomes whose function has been compromised by drugs.’ The precise function of ubiquitin and HSP 70 in lysosomes is not clear, although it presumably relates to the regulation of protein degradation. Certainly cells with a mutation of the ubiquitin activating enzyme E1 can no longer degrade proteins in lysosomes.9 In addition, ubiquitin and HSP 70 are useful markers of the lysosome system in both health and disease.
Ubiquitin-protein conjugates in health and disease Deposits of ubiquitin-protein conjugates are seen within the neuropil of the normal elderly human brain in numbers that increase with age. 10 11 These are nerve cell processes (neurites) packed with ubiquitin-immunoreactive lysosome-related dense ADDRESSES Departments of Biochemistry (Prof R. J Mayer, DSc, M. Landon, PhD, L Laszlo, PhD), Pathology (J Lowe, MRCPath), and Neurology (G. Lennox, BM), University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, UK. Correspondence to Prof R John Mayer
157
Fig 1-Dot-like
structures
immunohistochemistry (temporal lobe).
in
demonstrated by ubiquitin aged human cerebral cortex
(fig 1). Similar lysosome-related accumulations of ubiquitinated proteins are found in several pathological conditions. Dot-like structures are seen in the neuropil in mouse scrapie (an animal model of prion encephalopathy), together with coarser structures adjacent to neurons which resemble autophagic vacuoles .12 The dot-like structures develop very early after infection, at the time when abnormal prion protein first becomes detectable." Later on larger deposits of ubiquitinated proteins develop in structures resembling lysosomes, and in dystrophic neurites around amyloid plaques (fig 2). Identical structures are seen in the equivalent human prion diseases (Creutzfeldt-Jakob disease" and Gerstmann-Straussler-Scheinker syndrome 15). Similarly in Alzheimer disease deposits of ubiquitin-protein conjugates are seen within dystrophic neurites around senile plaques; these appear to occur in membranous and vesicular dense bodies resembling lysosomes. 16 Other characteristic elements of the pathology of Alzheimer disease also contain ubiquitinated proteins, such as the neurofibrillary tangles17-19 and areas of granulovacuolar degeneration (the latter again bearing some topographical resemblance to lysosomal structures2O). It has generally been assumed that these observations are epiphenomena, the persistence of ubiquinated proteins and their accumulation into lysosomal structures representing an appropriate and cytoprotective (if ultimately ineffective) response to the disease process. There is, however, now evidence to suggest that this response may itself be pathogenic and play a central part in causing neuronal damage. bodies
Fig 2-Lysosome-related multivesicular body in cerebral cortex of mouse with prion encephalopathy showing reactivity with ubiquitin immunogold electron microscopy.
disease the abnormal prion protein will be taken up into the lysosome-related system by some phagocytic process. This will inevitably incorporate portions of cell membrane (including normal prion protein) into endosomes; both normal and abnormal prion isoforms will then enter multivesicular bodies. In the acidic denaturing milieu, unfolded and part-fragmented prion protein molecules will be able to undergo the secondary structural interaction which is thought to result in the generation of further abnormal prion protein. Eventually a critical level of abnormal prion protein will accumulate, first disrupting the lysosomal membrane and then releasing hydrolases into the I
Lysosome-related organelles, prion encephalopathies, and Alzheimer disease Recent advances in identifying the prion protein (PrPC) and understanding its molecular genetics21,22 have shed little light upon the mechanisms which underlie its transformation into an abnormal isoform, its accumulation, and the subsequent development of spongiform pathology. The normal prion protein is a membrane protein found in a variety of cell types, including neurons.23 Cell culture studies of scrapie have shown that the abnormal isoform of prion protein appears within intracellular organelles24,25 which include lysosome-related structures.26 These are capable of partly truncating the protein27 and may provide an environment in which the abnormal isoform is generated from the normal prion protein. A hypothetical scheme for this is shown in fig 3. During natural or experimental infection with prion
Fig 3-Role of lysosome in biogenesis of abnormal prion protein. PrPc denotes normal prion protein, PrPsc its abnormal isoform
158
cell. Many of these enzymes retain activity at neutral pH and will cause neuronal damage. Ultimately, when most lysosomes in a neuron are overwhelmed by accumulated abnormal prion protein, the neuron will die and release abnormal prion protein to be taken up by other neurons through phagocytosis. This would lead to an exponential increase of abnormal prion protein culminating in neurodegeneration. In this context the lysosomal system is acting as the "bioreactor" for the formation of abnormal prion protein. This scheme is supported by pathological studies of murine scrapie, where immunogold electronmicroscopy has shown an accumulation of lysosome-related multivesicular, tubulovesicular and dense bodies containing hydrolases such as 0-glucuronidase as well as HSP 70, ubiquitin conjugates, and abnormal prion protein. All of these can be seen spilling out of the lysosome-related vesicles into areas of rarefaction which are thought to be the precursors of the larger areas of spongiform change which characterise the prion encephalopathies.18 Similar ubiquitin deposits and lysosomes can be seen immunohistochemically adjacent to spongiform lesions in Creutzfeldt-Jakob disease.29 A similar model can be constructed for Alzheimer disease. Here the amyloid precursor protein is thought to have a central role.3° It is concentrated in neuronal lysosomes in both normal and Alzheimer disease brain," 31 where it is subject to partial degradation.33,36 Again the acidic denaturing interior of lysosome-related structures provides an ideal environment for the transformation of the amyloid precursor protein into smaller P/A4 fragments and for the secondary structural interactions which are required for amyloid formation. In Alzheimer disease the fate of the bioreactor lysosomes may be slightly different; fusion of the lysosomes with the neuronal membrane would lead to the expulsion of their amyloid contents before neuronal death of extracellular amyloid, and the accumulation characteristically seen at the centre of senile plaques. An ejection mechanism of this kind is well-documented in other contexts-for example, in exporting transferrin receptors from maturing reticulocytes.34
New therapeutic targets? The nervous system, with its long-lived neurons, is vitally dependent on an effective lysosomal waste disposal system. Unlike other cell types, neurons cannot divide to replace cells that have died through the accumulation of indigestible material. Processing of proteins may become an increasing burden with ageing, and this accounts for the development of lysosome-related ubiquitinated dot-like structures in elderly neurons. These probably represent a common neuronal response to ageing and cell injury, reflecting activation of the lysosome-related system. Protein processing in the lysosome-related system, modulated by the cell stress proteins, may be crucial in
disease
states.
Modification of normal neuronal precursor
proteins and the accumulation or deposition of their abnormal products appears to be a central part of neurodegenerative diseases like Alzheimer disease and the prion encephalopathies. The lysosome system provides the only intracellular environment capable of performing this pathological processing, and the recent observations reviewed here suggest that it lies at the heart of the pathogenesis of these diseases. This raises the possibility that interference with protein processing in lysosomerelated organelles might confer therapeutic benefit. In Alzheimer disease it might be possible to block the
conversion of amyloid precursor protein to the P;A4 fragments and amyloid, so preventing deposition of the latter within and around neurons. In the prion encephalopathies it might be possible to prevent the rupture of lysosome-related bodies into neurons and halt the development of spongiform change. In both cases such interventions might usefully slow the progression of the disease. Drugs that influence lysosomal function already exist, as do transgenic models for at least the prion encephalopathies,35 which would allow this strategy to be rapidly put to the test. In the absence of any other effective treatment, we suggest that approaches to lysosomal intervention merit serious consideration. We thank the Well come Trust, Parkinson’s Disease Society, and Motor Neurone Disease Association for support of some of this work.
REFERENCES 1. Holzmann E. Lysosomes. New York: Plenum Press, 1989. 2. Chiang H-L, Terlecky S, Plant C, et al. A role for a 70 kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science 1989; 246: 282-84. 3. Lowe J, Mayer RJ. Ubiquitin, cell stress and diseases of the nervous system. Neuropathol Appl Neurobiol 1990; 16: 281-91. 4. Schwartz AL, Ciechanover A, Brandt RA, et al. Immunoelectron microscopic localization of ubiquitin in hepatoma cells. EMBO J 1988; 7: 2961-66. 5. Laszlo L, Doherty FJ, Osbom NU, et al. Ubiquitinated protein conjugates are specifically enriched in the lysosomal system of fibroblasts. FEBS Lett 1990; 261: 365-68. 6. Laszlo L, Tuckwell J, Self T, et al. The latent membrane protein-1 in Epstein-Barr virus-transformed lymphoblastoid cells is found with ubiquitin-protein conjugates and heat-shock protein 70 in lysosomes oriented around the microtubule organising centre. J Pathol 1991; 164: 203-14. 7. Laszlo L, Doherty FJ, Watson A, et al. Immunogold localisation of ubiquitin-protein conjugates in primary (azurophilic) granules of polymorphonuclear neutrophils. FEBS Lett 1991; 279: 175-78. 8. Doherty FJ, Osborn NU, Wassall JA, et al. Ubiquitin-protein conjugates accumulate in the lysosomal system of fibroblasts treated with cysteine proteinase inhibitors. Biochem J 1989; 263: 47-55. 9. Gropper R, Brandt RA, Elias S, et al. The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins. J Biol Chem 1991; 266: 3602-10. 10. Migheli A, Attanasio A, Pezzulo T, et al. Age-related ubiquitin deposits in dystrophic neurites: an immunoelectron microscopic study. Neuropathol Appl Neurobiol 1992; 121: 55-58. 11. Dickson DW, Wertkin A, Kress Y, et al. Ubiquitin immunoreactive structures in normal human brains. Lab Invest 1990; 63: 87-99. 12. Lowe J, McDermott H, Kenward N, et al. Ubiquitin conjugate immunoreactivity in the brains of scrapie infected mice. J Pathol 1990; 162: 61-66. 13. Lowe J, Fergusson J, Kenward N, et al. Immunoreactivity to ubiquitinprotein conjugates is present early in the process in the brains of scrapie infected mice. J Pathol (in pres 14. Suenaga T, Hirano A, Llena JF, et al. immunoreactivity in kuru plaques in Creutzfeld-Jakob disease Ing Neurol 1990; 28: 174-77. 15. Migheli A, Attanasio A, Vigliani MC, et al. Dystrophic neurites around amyloid plaques of human patients with Gerstmann-StrausslerScheinker disease contain ubiquitinated inclusions. Neurosci Lett 1991; 121: 55-58. 16. Dickson DW, Wertkin A, Mattiace LA, et al. Ubiquitin immunoelectron microscopy of dystrophic neurites in cerebellar senile plaques of Alzheimers disease. Acta Neuropathol 1990; 79: 486-93. 17. Mori H, Kondo J, Ihara Y. Ubiquitin is a component of paired helical filaments in Alzheimer’s disease. Science 1987; 235: 1641-44. 18. Perry G, Friedman R, Shaw G, et al. Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains. Proc Natl Acad Sci USA 1987; 84: 3033-36. 19. Cole GM, Timiras PS. Ubiquitin-protein conjugates in Alzheimer’s lesions. Neurosci Lett 1987; 79: 207-12. 20. Lowe J, Blanchard A, Morrell K, et al. Ubiquitin is a common factor in intermediate filament inclusion bodies of diverse type in man, including those of Parkinson’s disease, Pick’s disease, and Alzheimer’s disease, as well as Rosenthal fibres in cerebellar astrocytomas, cytoplasmic bodies in muscle, and Mallory bodies in alcoholic liver disease. J Pathol 1988; 155: 9-15. 21. Prusiner SB Molecular biology of prion diseases. Science 1991; 252: 1515-22.
159
Wright A, Goedert M, Hastie N. Beta amyloid resurrected. Nature 1991;
22. Brown P, Goldfarb LG, Gajdusek DC. The new biology of spongiform encephalopathy: infectious amyloidoses with a genetic twist. Lancet
30.
1991; 337: 1019-22. 23. Stahl N, Borchelt DR, Hsiao KK, et al. Glycolipid modification of the scrapie prion protein. Cell 1987; 51: 229-40. 24. Borchelt DR, Scott M, Taraboulos A, et al. Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. J Cell Biol 1990; 110: 743-52. 25. Taraboulos A, Serban D, Prusiner SB. Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells. J Cell Biol 1990;
31. Benowitz
110: 2117-32.
26. McKinley MP, Taraboulos A, Kenaga L, et al. Ultrastructural localisation of scrapie prions in cytoplasmic vesicles of infected cultured cells. Lab Invest 1991; 65: 622-30. 27. Caughey B, Raymond GJ, Ernst D, et al. N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistate state. J Virol 1991; 65: 6597-603. 28. Laszlo L, Lowe J, Self T, et al. Lysosomes are key organelles in the pathogenesis of prion encephalopathies. J Pathol 1992; 166: 333-41. 29. Ironside JW, Bell JE, McCardle L, et al. Neuronal and glial reactions in Creutzfeldt-Jakob disease. Neuropathol Appl Neurobiol 1992; 18: 295.
349: 653-54.
LI, Rodnguez W, Paskevich P, et al. The amyloid precursor protein is concentrated in neuronal lysosomes in normal and Alzheimer disease subjects. Exp Neurol 1989; 106: 237-50. 32. Kawai M, Cras P, Richey P, et al. Subcellular localisation of amyloid precursor protein in senile plaques of Alzheimer’s disease. Am J Pathol 1992; 140: 947-58. 33. Golde TE, Estus S, Younkin LH, et al. Processing of the amyloid precursor protein to potentially amyloidogenic derivatives. Science 1992; 255: 728-30. 34. David JQ, Dansereau D, Johnstone RM, et al. Selective externalization of an ATP-binding protein structurally related to clathrin uncoating ATPase heat shock protein in vesicles containing terminal transferrin receptors during reticulocyte maturation. J Biol Chem 1986; 261: 15368-71. 35. Hsaio
K, Scott M, Foster D, et al. Spontaneous neurodegeneration in transgenic mice with mutant prion protein. Science 1990; 250: 1587-60. 36. Haas C, Koo EH, Mellon A, et al. Targeting of cell-surface &bgr;-amyloid precursor protein to lysosomes: alternative processing into amyloidbearing fragments. Nature 1992; 357: 500-03.
HYPOTHESIS Primary aldosteronism: hypertension with a genetic basis
Exciting developments in knowledge of primary aldosteronism include description of new subtypes and elucidation of the genetic basis of one variety. Furthermore, relatively simple biochemical screening (aldosterone/renin ratio) has disclosed that primary aldosteronism is more common than previously
thought, by diagnosing patients
at
an
earlier,
normokalaemic stage. The mutant gene discovered in the glucocorticoid-suppressible variety (FHI) codes for an aldosterone biosynythetic enzyme normally controlled by angiotensin II, and now controlled by corticotropin. The zona fasciculata is hyperplastic and makes aldosterone and "hybrid steroids" 18-oxocortisol and 18-hydroxycortisol in excess, in response to ACTH but not to angiotensin II. Adrenal tumours have not yet been described in this condition. Aldosterone-producing adenomas (Conn’s syndrome) are also commonly composed of zona fasciculata-like cells, make "hybrid steroids" in excess and are very sensitive to ACTH but not to angiotensin II. We have described a new variety of is aldosterone-producing adenoma which responsive to angiotensin II (All-responsive APA), consists of at least 20% zona glomerulosa-like cells, and does not make "hybrid steroids" in excess. We have also described a new familial variety of primary aldosteronism that includes tumours and is not glucocorticoid-suppressible (FHII). We propose that primary aldosteronism is a spectrum of genetic diseases expressed as either hyperplasia or neoplasia, and that morphological and genetic diversity explains biochemical and clinical behaviour.
Earlier this year the genetic basis of a form of human was described for the first time.! It appears that a mutation in the genes (GYP11Bl and CYP11B2) coding for steroid biosynthetic enzymes that catalyse respectively cortisol production in the zona fasciculata under the control of corticotropin (ACTH) and aldosterone production in the zona glomerulosa under the control of angiotensin, resulting in a hybrid gene, is responsible. This new gene contains the promoter region of GYP 11 B and the coding region of C YP1 11 B2; it produces an enzyme that catalyses the formation of aldosterone but is very sensitive to ACTH. As a result, normal levels of ACTH (which maintain normal levels of cortisol) lead to excessive production of aldosterone with consequent hypertension and hypokalaemia. The phenotypic condition, known
hypertension
as variously glucocorticoid-suppressible hyperaldosteronism, glucocorticoid-remediable aldosteronism, and dexamethasone-suppressible hyperaldosteronism, was first described in 1966 by Sutherland et aF and is transmitted by a dominant mode of inheritance. It is considered rare, involves adrenocortical hyperplasia which may be nodular2but has never been reported to progress to tumour formation. Histological features suggest overactivity
of the zona fasciculata." Of 93 consecutive patients with primary aldosteronism diagnosed by us between 1970 and 1990, only 3 had glucocorticoid-suppressible hyperaldosteronism.5,6 In this condition, aldosterone production is very sensitive to ACTH but unresponsive to angiotensin II (upright posture
ADDRESS Endocrine-Hypertension Research Unit, University Department of Medicine, Greenslopes Hospital, Brisbane, Queensland 4120, Australia (Prof R. D. Gordon, FRACP, S. A. Klemm, PhD, T. J. Tunny, MSc, M. Stowasser, FRACP). Correspondence to Prof Richard D. Gordon.
low molecular weight heparin in patients undergoing total hip replacement. Thromb Haemostas 1988; 60: 407-11. 50. Samama M, Bernard P, Bonnardot JP, Combe-Tamzali S, Lanson Y, Tissot E. Low molecular weight heparin with unfractionated heparin in prevention of postoperative thrombosis. Br J Surg 1988; 75: 128-31. 51. Sasahara AA, Koppenhagen K, Häring R, Welzel D, Wolf H. Low molecular weight heparin plus dihydroergotamine for prophylaxis of postoperative deep vein thrombosis. Br J Surg 1986; 73: 697-700. 52. Verardi S, Casciani CU, Nicora E, et al. A multicentre study on LMW-heparin effectiveness in preventing postsurgical thrombosis. Int Angio 1988; 7 (suppl 3): 19-24. 53. Verardi S, Cortese F, Baroni B, Boffo V, Casciani CU, Palazinni E. Deep vein thrombosis prevention in surgical patients: effectiveness and safety
REVIEW
of
a new
low molecular
weight heparin. Curr Ther Res 1989;
46:
366-72.
Welzel D, Stringer MD, Hedges AR, et al. Fixed combinations of low molecular weight or unfractionated heparin plus dihydroergotamine in the prevention of postoperative deep vein thrombosis. Thromb Haemostas 1989; 62: 523 (abstr). 55. Cruickshank MK, Levine MN, Hirsh J, et al. An elevation of impedance plethysmography and 125-I-fibrinogen legscanning in patients following hip surgery. Thromb Haemostas 1989; 62: 830-36. 56. Laupacis A, Sackett DL, Roberts RS. An assessment of clinically useful measures of the consequences of treatment. N Engl J Med 1988; 318:
54.
1728-33. 57.
Light RS, Pillemer DB. Summing up: the science of reviewing research. Cambridge, Mass: Harvard University Press, 1984: 66-65.
ARTICLE processing in lysosomes: the new therapeutic target in neurodegenerative disease Protein
recognised feature of neurons is their large complement of lysosomes. Studies of the accumulation of the abnormal isoform of the prion protein (PrPSC) in the prion encephalopathies and the formation of &bgr;/A4 protein from its precursor in Alzheimer’s disease suggest that generation of these key proteins takes place in lysosome-related organelles. The release of hydrolytic enzymes from lysosomes may be a primary cause of neuronal damage. Although molecular genetic approaches have identified protein mutations central to the main cell disease, neurodegenerative biological observations are now beginning to unravel the intracellular pathways involved in the molecular pathogenesis of neurodegeneration: as a result, it is now appropriate to consider therapeutic manipulation of the lysosomal system as an approach to treatment. A little
Introduction Neurons do not replicate in adult life, so they need an efficient way of turning over proteins and dealing with any abnormal proteins. To this end they possess a very well-developed lysosome system. Evidence is accumulating to suggest that abortive attempts to degrade proteins within this system lie at the centre of the pathogenesis of some of the major neurodegenerative diseases of man. These include Alzheimer disease and the prion encephalopathies such as Creutzfeldt-Jakob disease where abnormal amyloid (&bgr;/A4) and prion (PrPSC)proteins, respectively, are deposited in and around neurons. This in turn opens up the possibility of new therapeutic strategies, aimed at altering lysosomal
protein processing.
Lysosome system the most familiar part of the large system Lysosomes of acid-containing vesicles that enable cells to digest unwanted material. They are characterised by specific hydro lases (eg, &bgr;-glucuronidase) which are most active at are
low pH. Other components of this acidic vesicle system include endosomes (vesicles formed after membrane intemalisation during receptor-mediated endocytosis), multivesicular and tubulovesicular bodies (which may form by the surface invagination of endosomes), autophagic vacuoles (formed within cells to isolate unwanted organelles), and nascent hydrolase-containing vesicles derived from the protein-packaging Golgi apparatus.l Recent evidence suggests that the lysosome system interacts closely with cell stress proteins. Cell stress proteins-also known as heat-shock proteins (HSP) after one form of cell stress used in early experiments-are highly conserved and have roles in normal cell activity as well as in the protective response to cell damage. They include ubiquitin, a central co-factor in protein degradation, and HSP 70,2 which acts as a molecular "chaperone", facilitating the folding and transport of proteins across different compartments within the cell.3 Initially thought of as cytosolic proteins, both are also found within lysosome related organelles. Immunogold electronmicroscopy has shown that normal lysosomes contain both free ubiquitin’ and ubiquitin-protein conjugates5-7 and that these conjugates accumulate excessively in lysosomes whose function has been compromised by drugs.’ The precise function of ubiquitin and HSP 70 in lysosomes is not clear, although it presumably relates to the regulation of protein degradation. Certainly cells with a mutation of the ubiquitin activating enzyme E1 can no longer degrade proteins in lysosomes.9 In addition, ubiquitin and HSP 70 are useful markers of the lysosome system in both health and disease.
Ubiquitin-protein conjugates in health and disease Deposits of ubiquitin-protein conjugates are seen within the neuropil of the normal elderly human brain in numbers that increase with age. 10 11 These are nerve cell processes (neurites) packed with ubiquitin-immunoreactive lysosome-related dense ADDRESSES Departments of Biochemistry (Prof R. J Mayer, DSc, M. Landon, PhD, L Laszlo, PhD), Pathology (J Lowe, MRCPath), and Neurology (G. Lennox, BM), University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, UK. Correspondence to Prof R John Mayer
157
Fig 1-Dot-like
structures
immunohistochemistry (temporal lobe).
in
demonstrated by ubiquitin aged human cerebral cortex
(fig 1). Similar lysosome-related accumulations of ubiquitinated proteins are found in several pathological conditions. Dot-like structures are seen in the neuropil in mouse scrapie (an animal model of prion encephalopathy), together with coarser structures adjacent to neurons which resemble autophagic vacuoles .12 The dot-like structures develop very early after infection, at the time when abnormal prion protein first becomes detectable." Later on larger deposits of ubiquitinated proteins develop in structures resembling lysosomes, and in dystrophic neurites around amyloid plaques (fig 2). Identical structures are seen in the equivalent human prion diseases (Creutzfeldt-Jakob disease" and Gerstmann-Straussler-Scheinker syndrome 15). Similarly in Alzheimer disease deposits of ubiquitin-protein conjugates are seen within dystrophic neurites around senile plaques; these appear to occur in membranous and vesicular dense bodies resembling lysosomes. 16 Other characteristic elements of the pathology of Alzheimer disease also contain ubiquitinated proteins, such as the neurofibrillary tangles17-19 and areas of granulovacuolar degeneration (the latter again bearing some topographical resemblance to lysosomal structures2O). It has generally been assumed that these observations are epiphenomena, the persistence of ubiquinated proteins and their accumulation into lysosomal structures representing an appropriate and cytoprotective (if ultimately ineffective) response to the disease process. There is, however, now evidence to suggest that this response may itself be pathogenic and play a central part in causing neuronal damage. bodies
Fig 2-Lysosome-related multivesicular body in cerebral cortex of mouse with prion encephalopathy showing reactivity with ubiquitin immunogold electron microscopy.
disease the abnormal prion protein will be taken up into the lysosome-related system by some phagocytic process. This will inevitably incorporate portions of cell membrane (including normal prion protein) into endosomes; both normal and abnormal prion isoforms will then enter multivesicular bodies. In the acidic denaturing milieu, unfolded and part-fragmented prion protein molecules will be able to undergo the secondary structural interaction which is thought to result in the generation of further abnormal prion protein. Eventually a critical level of abnormal prion protein will accumulate, first disrupting the lysosomal membrane and then releasing hydrolases into the I
Lysosome-related organelles, prion encephalopathies, and Alzheimer disease Recent advances in identifying the prion protein (PrPC) and understanding its molecular genetics21,22 have shed little light upon the mechanisms which underlie its transformation into an abnormal isoform, its accumulation, and the subsequent development of spongiform pathology. The normal prion protein is a membrane protein found in a variety of cell types, including neurons.23 Cell culture studies of scrapie have shown that the abnormal isoform of prion protein appears within intracellular organelles24,25 which include lysosome-related structures.26 These are capable of partly truncating the protein27 and may provide an environment in which the abnormal isoform is generated from the normal prion protein. A hypothetical scheme for this is shown in fig 3. During natural or experimental infection with prion
Fig 3-Role of lysosome in biogenesis of abnormal prion protein. PrPc denotes normal prion protein, PrPsc its abnormal isoform
158
cell. Many of these enzymes retain activity at neutral pH and will cause neuronal damage. Ultimately, when most lysosomes in a neuron are overwhelmed by accumulated abnormal prion protein, the neuron will die and release abnormal prion protein to be taken up by other neurons through phagocytosis. This would lead to an exponential increase of abnormal prion protein culminating in neurodegeneration. In this context the lysosomal system is acting as the "bioreactor" for the formation of abnormal prion protein. This scheme is supported by pathological studies of murine scrapie, where immunogold electronmicroscopy has shown an accumulation of lysosome-related multivesicular, tubulovesicular and dense bodies containing hydrolases such as 0-glucuronidase as well as HSP 70, ubiquitin conjugates, and abnormal prion protein. All of these can be seen spilling out of the lysosome-related vesicles into areas of rarefaction which are thought to be the precursors of the larger areas of spongiform change which characterise the prion encephalopathies.18 Similar ubiquitin deposits and lysosomes can be seen immunohistochemically adjacent to spongiform lesions in Creutzfeldt-Jakob disease.29 A similar model can be constructed for Alzheimer disease. Here the amyloid precursor protein is thought to have a central role.3° It is concentrated in neuronal lysosomes in both normal and Alzheimer disease brain," 31 where it is subject to partial degradation.33,36 Again the acidic denaturing interior of lysosome-related structures provides an ideal environment for the transformation of the amyloid precursor protein into smaller P/A4 fragments and for the secondary structural interactions which are required for amyloid formation. In Alzheimer disease the fate of the bioreactor lysosomes may be slightly different; fusion of the lysosomes with the neuronal membrane would lead to the expulsion of their amyloid contents before neuronal death of extracellular amyloid, and the accumulation characteristically seen at the centre of senile plaques. An ejection mechanism of this kind is well-documented in other contexts-for example, in exporting transferrin receptors from maturing reticulocytes.34
New therapeutic targets? The nervous system, with its long-lived neurons, is vitally dependent on an effective lysosomal waste disposal system. Unlike other cell types, neurons cannot divide to replace cells that have died through the accumulation of indigestible material. Processing of proteins may become an increasing burden with ageing, and this accounts for the development of lysosome-related ubiquitinated dot-like structures in elderly neurons. These probably represent a common neuronal response to ageing and cell injury, reflecting activation of the lysosome-related system. Protein processing in the lysosome-related system, modulated by the cell stress proteins, may be crucial in
disease
states.
Modification of normal neuronal precursor
proteins and the accumulation or deposition of their abnormal products appears to be a central part of neurodegenerative diseases like Alzheimer disease and the prion encephalopathies. The lysosome system provides the only intracellular environment capable of performing this pathological processing, and the recent observations reviewed here suggest that it lies at the heart of the pathogenesis of these diseases. This raises the possibility that interference with protein processing in lysosomerelated organelles might confer therapeutic benefit. In Alzheimer disease it might be possible to block the
conversion of amyloid precursor protein to the P;A4 fragments and amyloid, so preventing deposition of the latter within and around neurons. In the prion encephalopathies it might be possible to prevent the rupture of lysosome-related bodies into neurons and halt the development of spongiform change. In both cases such interventions might usefully slow the progression of the disease. Drugs that influence lysosomal function already exist, as do transgenic models for at least the prion encephalopathies,35 which would allow this strategy to be rapidly put to the test. In the absence of any other effective treatment, we suggest that approaches to lysosomal intervention merit serious consideration. We thank the Well come Trust, Parkinson’s Disease Society, and Motor Neurone Disease Association for support of some of this work.
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HYPOTHESIS Primary aldosteronism: hypertension with a genetic basis
Exciting developments in knowledge of primary aldosteronism include description of new subtypes and elucidation of the genetic basis of one variety. Furthermore, relatively simple biochemical screening (aldosterone/renin ratio) has disclosed that primary aldosteronism is more common than previously
thought, by diagnosing patients
at
an
earlier,
normokalaemic stage. The mutant gene discovered in the glucocorticoid-suppressible variety (FHI) codes for an aldosterone biosynythetic enzyme normally controlled by angiotensin II, and now controlled by corticotropin. The zona fasciculata is hyperplastic and makes aldosterone and "hybrid steroids" 18-oxocortisol and 18-hydroxycortisol in excess, in response to ACTH but not to angiotensin II. Adrenal tumours have not yet been described in this condition. Aldosterone-producing adenomas (Conn’s syndrome) are also commonly composed of zona fasciculata-like cells, make "hybrid steroids" in excess and are very sensitive to ACTH but not to angiotensin II. We have described a new variety of is aldosterone-producing adenoma which responsive to angiotensin II (All-responsive APA), consists of at least 20% zona glomerulosa-like cells, and does not make "hybrid steroids" in excess. We have also described a new familial variety of primary aldosteronism that includes tumours and is not glucocorticoid-suppressible (FHII). We propose that primary aldosteronism is a spectrum of genetic diseases expressed as either hyperplasia or neoplasia, and that morphological and genetic diversity explains biochemical and clinical behaviour.
Earlier this year the genetic basis of a form of human was described for the first time.! It appears that a mutation in the genes (GYP11Bl and CYP11B2) coding for steroid biosynthetic enzymes that catalyse respectively cortisol production in the zona fasciculata under the control of corticotropin (ACTH) and aldosterone production in the zona glomerulosa under the control of angiotensin, resulting in a hybrid gene, is responsible. This new gene contains the promoter region of GYP 11 B and the coding region of C YP1 11 B2; it produces an enzyme that catalyses the formation of aldosterone but is very sensitive to ACTH. As a result, normal levels of ACTH (which maintain normal levels of cortisol) lead to excessive production of aldosterone with consequent hypertension and hypokalaemia. The phenotypic condition, known
hypertension
as variously glucocorticoid-suppressible hyperaldosteronism, glucocorticoid-remediable aldosteronism, and dexamethasone-suppressible hyperaldosteronism, was first described in 1966 by Sutherland et aF and is transmitted by a dominant mode of inheritance. It is considered rare, involves adrenocortical hyperplasia which may be nodular2but has never been reported to progress to tumour formation. Histological features suggest overactivity
of the zona fasciculata." Of 93 consecutive patients with primary aldosteronism diagnosed by us between 1970 and 1990, only 3 had glucocorticoid-suppressible hyperaldosteronism.5,6 In this condition, aldosterone production is very sensitive to ACTH but unresponsive to angiotensin II (upright posture
ADDRESS Endocrine-Hypertension Research Unit, University Department of Medicine, Greenslopes Hospital, Brisbane, Queensland 4120, Australia (Prof R. D. Gordon, FRACP, S. A. Klemm, PhD, T. J. Tunny, MSc, M. Stowasser, FRACP). Correspondence to Prof Richard D. Gordon.
