Nature Medicine 10, S10–S17 (2004)

Protein aggregation and neurodegenerative disease

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and prion diseases are increasingly being realized to have common cellular and molecular mechanisms including protein aggregation and inclusion body formation. The aggregates usually consist of fibers containing misfolded protein with a -sheet conformation, termed amyloid. There is partial but not perfect overlap among the cells in which abnormal proteins are deposited and the cells that degenerate. The most likely explanation is that inclusions and other visible protein aggregates represent an end stage of a molecular cascade of several steps, and that earlier steps in the cascade may be more directly tied to pathogenesis than the inclusions themselves. For several diseases, genetic variants assist in explaining the pathogenesis of the more common sporadic forms and developing mouse and other models. There is now increased understanding of the pathways involved in protein aggregation, and some recent clues have emerged as to the molecular mechanisms of cellular toxicity. These are leading to approaches toward rational therapeutics.

Christopher A Ross¹ & Michelle A Poirier<sup>2

1</sup> Christopher A. Ross is in the Division of Neurobiology, Department of Psychiatry, and Departments of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Ross Research Building, Room 618, 720 Rutland Avenue, Baltimore, Maryland 21205, USA. caross@jhu.edu

² Michelle A. Poirier is in the Division of Neurobiology, Department of Psychiatry, Johns Hopkins University School of Medicine, Ross Research Building, Room 618, 720 Rutland Avenue, Baltimore, Maryland 21205, USA.

Published online: 1 July 2004
doi:10.1038/nm1066

Fig. 1
		Figure 1 | Characteristic neurodegenerative disease neuropathological lesions involve deposition of abnormal proteins, which can be intranuclear, cytoplasmic or extracellular.

Table 1
		Table 1 | Neurodegenerative diseases: proteins and pathology

Huntington's disease. HD is a progressive neurodegenerative disorder caused by expansion of a CAG repeat coding for polyglutamine in the N terminus of the huntingtin protein. Because it is caused by a mutation in a single gene, HD has emerged as a model for studying neurodegenerative disease pathogenesis. There is a remarkable threshold effect, in that polyglutamine stretches of 36 in huntingtin cause disease, whereas 35 do not. Within the expanded range, longer repeats cause earlier onset. There is a striking correlation between the threshold for aggregation in vitro and the threshold for disease in humans, consistent with the idea that aggregation is related to pathogenesis^{10, 11}. Inclusions containing huntingtin are present in regions of the brain that degenerate. However, the neurons with inclusions do not correspond exactly to the neurons that degenerate. For instance, inclusions are present in the striatum, which is most affected¹², but they are more enriched in populations of large interneurons, which are spared, than in medium spiny projection neurons, which are selectively lost¹³. There is a good correlation, however, between the length of the CAG repeat and the density of inclusions^{12, 13, 14, 15, 16}.

Huntingtin aggregates can be labeled with antibodies to the N terminus of huntingtin or antibodies to ubiquitin, a marker for misfolded proteins, and a signal for degradation by the proteasome. Proteasomes may have difficulty digesting them, however, leading to their accumulation¹⁷. The aggregates contain fibers and appear to have -sheet structure characteristic of amyloid¹⁰, although there is controversy about whether they bind dyes that intercalate into -SHEETS, as is a characteristic of amyloid. Other proteins, such as Creb binding protein (CBP; discussed later) containing polyglutamine may be recruited into huntingtin aggregates^{18, 19}.

Alzheimer's disease and tauopathies. AD is a late-onset dementing illness, with progressive loss of memory, task performance, speech, and recognition of people and objects. There is degeneration of neurons (particularly in the basal forebrain and hippocampus), but at least as important for pathogenesis may be synaptic pathology and altered neuronal connections^{23, 24}. AD involves two major kinds of protein aggregates. Extracellular aggregates known as neuritic plaques have as their major constituent the A peptide, which is derived from proteolytic processing of the amyloid precursor protein (APP). The A-containing aggregates have -sheet structure and Congo red and thioflavin-T reactivity characteristic of amyloid²⁵. There are also intracellular aggregates of the microtubule-associated protein tau, called neurofibrillary tangles. The pathogenesis of AD has been greatly clarified by the identification of genetic mutations responsible for rare familial forms of the disease. These mutations are in APP itself and also in the presenilins, which are involved with the cleavage of APP (refs. 26,27). In addition, tauopathies such as fronto-temporal dementia with parkinsonism can be caused by mutations in the tau protein^{28, 29}.

Parkinson's disease. PD is characterized by resting tremor, rigidity, slow movements and other features such as postural and autonomic instability. It is caused by degeneration of dopaminergic neurons in the substantia nigra of the midbrain and other monoaminergic neurons in the brain stem³⁰. The discovery of several genes in which mutations cause early-onset forms of PD has greatly accelerated research progress³¹. Point mutations or increased gene dosage of the -synuclein gene cause autosomal dominant PD via a gain-of-function mechanism. Recessive early-onset PD can be caused by mutations in the genes encoding parkin, DJ-1 or PINK1³², presumably by a loss-of-function mechanism. The pathological hallmark of adult-onset PD is the Lewy body, an inclusion body found in the cytoplasm of neurons, often near the nucleus. Lewy bodies are densest in the substantial nigra but can also be present in monoaminergic, cerebral cortical and other neurons. There are also aggregates in neurites, which are referred to as Lewy neurites. A major constituent of Lewy bodies is aggregated -synuclein protein. Lewy bodies can also be labeled for ubiquitin, a synuclein interactor termed synphilin-1, proteasome proteins, and cytoskeletal and other proteins.

Amyloid fibrils are filamentous structures with a width of 10 nm and a length of 0.1–10 m. A defining feature, originally revealed by X-RAY FIBER DIFFRACTION ANALYSIS^{41, 42}, is the presence of cross- structure. In this structural motif, ribbonlike -sheets are formed by -strands running nearly perpendicular to the long axis of the fibril and hydrogen bonds that run nearly parallel to the long axis.

The most extensively characterized amyloid fibril is that formed from the -amyloid (A) peptide implicated in AD. Using SOLID-STATE NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY, the in-register, parallel -sheet organization of fibrils formed by A_10–35, a fragment of the full-length 42-residue A peptide, was first described⁴³. It was subsequently found that full-length A_1–42 forms -sheets with the same registry and orientation⁴⁴. Using ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY, a similar structural model was obtained for A peptide and A₄₀ (ref. 45).

A similar analysis of fibrils formed by -synuclein found an in-register, parallel -sheet organization⁴⁶. The core structure of A, -synuclein and polyglutamine aggregates appears to involve both -strands and -turns^{47, 48, 49, 50}. Recent data suggest that the structure of polyglutamine aggregates may involve a compact -sheet with interspersed -turns every nine glutamines^{50, 51, 52}. Thus a -sheet plus -turn structure may be a common form of neurodegenerative disase-related amyloid (see Fig. 2).

Fig. 2
		Figure 2 | -sheet, -turn models for expanded polyglutamine and A amyloid suggest commonalities in amyloid structure in different neurodegenerative diseases.

Consistent with a common structure, conformation-specific antibodies can bind to the amyloid fibril state of the A peptide but not to its soluble monomeric state. They also bind to amyloid fibrils and amyloid-like aggregates derived from other proteins of unrelated sequence including polyglutamine, but not to non-native globular protein aggregates such as collagen, gelatin or elastin⁵³. Thus, whereas there are still many unknowns regarding the detailed structure of amyloid and particularly regarding its assembly, there seem to be considerable similarities among the structures of different kinds of disease-related amyloid.

The initiation of misfolding in a particular cell may be a stochastic event, with a constant risk over the life of the individual⁵⁵. Amyloid formation may proceed via a process of 'seeded polymerization'^{56, 57, 58, 59}. The likelihood of aggregation could be increased by increasing protin concentration. This can be caused by genetic dosage alterations. For instance, familial PD can be caused by triplication at the -synuclein locus⁶⁰. Early deposition of A plaques occurs in individuals with Down's syndrome, who carry an extra copy of the APP locus on chromosome 21. Polymorphisms in promoter sites of disease-associated genes may increase transcription and thus protein amounts, increasing the risk for neurodegenerative disease⁶¹. In the case of protein-coding mutations, the altered primary structure presumably makes the protein more prone to aggregate. For polyglutamine proteins, there is a very clear correlation between the expansion of the polyglutamine stretch and the aggregation of polyglutamine itself.

Covalent modifications of proteins may facilitate aggregation. Sporadic neurodegenerative diseases are generally associated with aging, which is accompanied by oxidative modifications of proteins. Oxidative modification of -synuclein via dopamine adducts may facilitate aggregation⁶². Aging may also decrease the ability of the cell to clear misfolded proteins. Nitration of -synuclein has also been described⁶³, although whether this is an early or later event is not certain.

Another important covalent promoter of aggregation is phosphorylation. -Synuclein purified from Lewy bodies is extensively phosphorylated on Ser129 (refs. 64, 65, 66, 67), and experiments in cell culture suggest that Ser129 phosphorylation of -synuclein strongly modulates interactions between -synuclein and synphilin-1, and formation of inclusions. Thus, phosphorylation at Ser129 may have a role in the formation of Lewy bodies in PD.

Proteolytic cleavage may have a role in several of the neurodegenerative diseases, including AD. A is generated by the sequential action of -secretase and -secretase^{26, 27}. By contrast, APP can be cleaved normally into a non-amyloidogenic peptide by the combination of -secretase and -secretase. When APP is intact, it has very little tendency to aggregate, but the small cleavage product A has a strong tendency to aggregate. The cleavage site at which -secretase acts can vary by several amino acids, and A₄₀ is less toxic, and also aggregates less, than A₄₂.

A role for proteolytic cleavage in PD pathogenesis is less well established. Lewy bodies contain both N-terminal and C-terminal epitopes of -synuclein, indicating the presence of full-length protein. There may be also be truncated species, however. Recent observations of a transgenic mouse model of PD suggest the existence of several truncated species of -synuclein protein, enriched in the insoluble fraction⁷⁹. It is conceivable that these could initiate or facilitate the aggregation process.

A aggregation intermediates and toxicity. Several soluble oligomeric intermediates (larger than dimers) of A peptide variants have been described independently by several different groups. One researcher proposed that A₄₂ and the shorter 1–40 fragment form a 'micelle' structure in solution⁸². Another group identified spheroidal structures by ATOMIC FORCE MICROSCOPY (AFM) and referred to them as A 'protofibrils' (ref. 83). Finally, a third group described a globular intermediate for A₄₂ and gave it the name ADDL, for A-derived diffusable ligand⁸⁴. For simplicity, all of these species may be termed globular or oligomeric intermediates.

Chainlike fibrils have also been detected by AFM^{85, 86} and electron microscopy (EM)⁸⁷ for A variants. These species, referred to as protofibrils, often have a curvilinear morphology, are 4 nm in height by AFM and range between 6 and 10 nm in diameter by EM. Protofibrils are shorter than mature fibers, with a length range between 5 and 160 nm. Although the pathway of assembly is not certain, it seems that globular intermediates may polymerize further to form protofibrils⁸⁸. The term protofibril may best be reserved for small species with an early fibril-like morphology. Protofibrils then may assemble into protofilaments and finally mature fibers (Fig. 3).

Fig. 3
		Figure 3 | Flowchart for therapeutic intervention in a hypothetical several-step pathway of protein aggregation.

Although neuritic plaques are a hallmark of AD, there is a poor correlation between plaque density in human postmortem material and antemortem cognitive deficits⁸⁹. Soluble A intermediates have been observed in human postmortem material^{90, 91}. Toxicity in vitro has been described for both globular and protofibrillar intermediates^{84, 88}. Injection of purified A monomers and spheroids into rat hippocampus⁹² in vivo caused a block in long-term potentiation, substantiating a role for A aggregation intermediates in AD neurotoxicity.

Intermediates in -synuclein aggregation. Several different aggregation intermediates with size and morphology similar to those for A have been described for -synuclein. The pathway of assembly for intermediate forms of -synuclein may be complex, with globular and ringlike forms in addition to curvilinear protofibrils⁹³. Polyunsaturated fatty acids were reported to promote oligomerization, suggesting that -synuclein may aggregate via an interaction with cell membranes⁹⁴. One proposed mechanism of toxicity is the formation of pores by ringlike intermediates⁹³, although this idea is based on in vitro studies with recombinant protein.

Commonalities among soluble oligomeric intermediate species. As described earlier, aggregation intermediates have been widely observed in many of the neurodegenerative diseases. Recently, an antibody has been generated that reacts with oligomeric, but not with monomeric or fibrillar forms of polyglutamine, A, -synuclein and prion protein¹⁰⁰. This antibody recognized material in postmortem AD brain tissue that was distinct from plaques, and that blocked cell toxicity by A, -synuclein and polyglutamine. The actual mechanism for this block in toxicity is uncertain, because polyglutamine and -synuclein interact intracellularly whereas A interacts extracellularly. Nevertheless, it is tempting to speculate that a common structure of soluble nonfibrillar intermediates exists for all of these molecules, and that there may be common mechanisms of pathogenesis.

Other therapeutic interventions might directly reduce the level of abnormal protein within the cell, for instance using RNA interference¹¹¹, although its delivery would have to overcome formidable barriers of entry across the blood-brain barrier and access to neurons in the relevant region of the brain. For a disease such as PD (because the substantia nigra is relatively small), viral vectors could be directly injected. In patients with the dominant familial diseases, such as PD, ALS and AD, in which there are point mutations, it may be feasible to inactivate the mutant allele selectively¹¹². Another approach, at least for diseases involving extracellular aggregates, is to use antibodies. Immunization approaches to A have been tried with considerable success in animal models, but with side effects including encephalitis in humans^{113, 114, 115}.

Small molecules, which could be developed as drugs, may be able to target the protein misfolding pathway. Congo red binds to proteins with -sheet structure and may alter the protein misfolding pathway^{77, 80} and reduce toxicity in vivo⁹⁶. Chemical chaperones may be developed for blocking protein aggregation. The disaccharide trehalose¹¹⁶ has recently had some success for polyglutamine disease, although at high concentration.

Small-molecule agents are being developed to inhibit aggregation of A^{117, 118, 119}, -synuclein⁷⁹ and prions^{120, 121}. Small molecules can inhibit polyglutamine aggregation in vitro^{122, 123}. This has led to the development of an automated small-molecule screen for in vitro inhibitors of polyglutamine aggregation.

Another approach involves identifying specific pathogenic mechanisms for individual diseases and developing targeted therapy. Proteolytic cleavage is an especially attractive therapeutic target, because proteolytic enzymes may be amenable to the development of high-potency small-molecule inhibitors. This is a major strategy for AD, targeting both -secretase and -secretase^{27, 124}. There is great hope that better understanding of the pathogenic pathways will lead to rational therapeutics.

Acknowledgements

Supported by NINDS NS16375, NS38144, NS34172, NS38377, the Huntington's Disease Society of America, the Hereditary Disease Foundation, and the High-Q Foundation. We thank the anonymous reviewers for their comments and suggestions. JCT is supported by NINDS NS16375, NS38377 and NIA AG05146.

Competing interests statement

The authors declare that they have no competing financial interests.

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