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The Lancet, Vol. 340; July 18, 1992
REVIEW ARTICLE

Protein processing in lysosomes: the new therapeutic target in neurodegenerative disease

R. JOHN MAYER, MICHAEL LANDON, LAJOS LASZLO, GRAHAM LENNOX, JAMES LOWE

 

A little 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 β/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 neurodegenerative disease, cell 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.

Lancet 1992; 340:156-59

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 (β/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

Lysosomes are the most familiar part of the large system of acid-containing vesicles that enable cells to digest unwanted material. They are characterised by specific hydrolases (eg, (β-glucuronidase) which are most active at low pH. Other components of this acidic vesicle system include endosomes (vesicles formed after membrane internalisation 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.1

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 ubiquitin4 and ubiquitin-protein conjugates5-7 and that these conjugates accumulate excessively in lysosomes whose function has been compromised by drugs.8 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 El 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-immnunoreactive lysosome-related dense bodies (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.13 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 disease14 and Gerstmann-Straussler-Scheinker syndrome15).

Figure 1

Figure 2

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 structures20).

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.

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.

Figure 3

During natural or experimental infection with prion disease the abnormal prion protein will be taken up into the lysosome-related system by some phagocytic process. This will inevitably in 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 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 β-glucuronidase as well as HSP 70, ubiquitin conjugates, and abnormal priori 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.28 Similar ubiquitin deposits and lysosomes can be seen in immunohistochemically adjacent to spongiform lesions in Creutzfeldt disease.29

A similar model can be constructed for Alzheimer disease. Here the amyloid precursor protein is thought to have a central role.30 It is concentrated in neuronal lysosomes in both normal and Alzheimer disease brain,31,32 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 β/A 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 and the accumulation of extracellular amyloid, 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 lysosome related organelles might confer therapeutic benefit. In Alzheimer disease it might be possible to block the conversion of amyloid precursor protein to the β/A4 fragments and amyloid, so preventing deposition of the latter within and around neurons. In the priori 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 Wellcome Trust, Parkinson’s Disease Society, and Motor Neurone Disease Association for support of some of this work.

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3. Lowe J, Mayer RJ. Ubiquitin, cell stress and diseases of the nervous system. Neuropothol Appl Neurobiol 1990; 16: 281-91.

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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.

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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.


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