The carbonyl scavengers aminoguanidine and tenilsetam protect against the neurotoxic effects of methylglyoxal

Advanced glycation end products (AGEs) have been identified in age-related intracellular protein deposits of Alzheimer's disease (amyloid plaques and neurofibrillary tangles) and Parkinson disease (Lewy bodies), suggesting that these protein deposits have been exposed to AGE precursors such as the reactive dicarbonyl compound methylglyoxal. In ageing tissue and under diabetic pseudohypoxia, intracellular methylglyoxal levels rise through an impairment of triosephosphate utilization. Furthermore, methylglyoxal detoxification is impaired when reduced glutathione levels are low, conditions, which have all been described in Alzheimer's disease. However, there is less known about the toxicity of methylglyoxal, particularly about therapeutic strategies to scavenge such dicarbonyl compounds and attenuate their toxicity. In our study, extracellularly applied methylglyoxal was shown to be toxic to human neuroblastoma cells in a dose-dependent manner above concentrations of 150 microM with a LD50 of approximately 1.25 mM. Pre-incubation of methylglyoxal with a variety of carbonyl scavengers such as aminoguanidine or tenilsetam and the thiol antioxidant lipoic acid significantly reduced its toxicity. In summary, carbonyl scavengers might offer a promising therapeutic strategy to reduce the neurotoxicity of reactive carbonyl compounds, providing a potential benefit for patients with age-related neurodegenerative diseases.     

Ultrastructural localization of complement membrane attack complex (MAC)-like immunoreactivity in brains of patients with Alzheimer's disease

The membrane attack complex (MAC) of complement, also known as C5b-9, was localized in Alzheimer's disease (AD) brain by immunoelectron microscopy using a monoclonal antibody to a neoantigenic epitope of soluble C5b-9 (SC5b-9). Immunopositivity was detected in association with lamellated bodies in the neuronal cytoplasm, lipofuscin granules, lysosomes and neurofibrillary tangles (NFTs). Such intracellular localization of MAC-like immunoreactive (MAC-LI) staining suggests that neurons remove membrane-inserted MAC fragments by endocytosis. These endocytosed membrane fragments then proceed by retrograde transport to the perikaryon for lysosomal degradation. Attachment to the abnormal cytoskeletal proteins found in neurofibrillary tangles also occurs. The results provide further evidence that complement-mediated injury of neurons plays a part in the pathophysiology of AD.     

Capillary and arterial cerebral amyloid angiopathy in Alzheimer's disease: defining the perivascular route for the elimination of amyloid beta from the human brain

Accumulation of amyloid beta (Abeta) in the extracellular spaces of the cerebral cortex and in blood vessel walls as cerebral amyloid angiopathy is a characteristic of Alzheimer's disease (AD) and the ageing human brain. Studies in animals suggest that Abeta is eliminated from the brain either directly into the blood or along perivascular interstitial fluid drainage channels. The aim of the present study is to define the perivascular route for the drainage of Abeta from the human brain. Smears and paraffin sections of post-mortem cortical tissue from 17 cases of AD and from two controls were stained with thioflavin and for Abeta by immunohistochemistry. Histology and confocal microscopy showed that deposits of Abeta in the cortical parenchyma were continuous with Abeta in capillary walls but Abeta in artery walls was not in continuity with Abeta in brain parenchyma. Quantitative studies supported these observations. The results of this study suggest that when Abeta is eliminated from the extracellular spaces of the human brain by the perivascular route, it enters pericapillary spaces and from there drains along the walls of cortical arteries to leptomeningeal arteries. Factors such as overproduction of Abeta, entrapment of Abeta in drainage pathways and poor drainage of Abeta due to functional changes in ageing arteries might result in the failure of elimination of Abeta from the ageing brain and play a major role in the pathogenesis of AD. Such factors might affect therapies for AD that entail administration of anti-Abeta antibodies to eliminate Abeta from the human brain.     

Ubiquitin immunocytochemistry in human spongiform encephalopathies

The distribution of ubiquitin was studied by immunocytochemistry in eight cases of human spongiform encephalopathy and compared with the findings in seven age-and sex-matched cases of Alzheimer's disease and six non-demented control cases. The results were also compared with the immunocytochemical distribution of prion protein and the lysosomal aspartic protease cathepsin D. In the human spongiform encephalopathies, ubiquitin immunoreactivity was found in a punctate distribution at the periphery of prion protein amyloid plaques and in a finely granular pattern in the neuropil around and within areas of spongiform change. Cortical nerve cells contained scanty ubiquitinated dot-like inclusions, and occasional microglia around the areas of spongiform change also gave a positive staining reaction for ubiquitin, as did multiple irregular thread-like structures in the neuropil and white matter. The ubiquitin-containing structures at the plaque periphery in human spongiform encephalopathies resemble the neuritic processes at the periphery of the senile plaque in Alzheimer's disease. The granular positivity for ubiquitin associated with areas of spongiform change closely resembles the pattern of immunostaining seen with the antibodies to the prion protein and cathepsin D, consistent with the reported accumulation of ubiquitinated proteins and prion protein in lysosomes in the murine scrapie model. Further studies are required to investigate the role of lysosomes in this group of disorders, and to study the localization of other cell stress proteins and prion protein in spongiform encephalopathies.     

Widespread serum amyloid P immunoreactivity in cortical amyloid deposits and the neurofibrillary pathology of Alzheimer's disease and other degenerative disorders

Amyloid P (AP) component is present in all types of systemic amyloid deposits. Recently, it has been shown to be also present in cerebral amyloid lesions of Alzheimer's disease (AD). In this study, we used immunocytochemical methods to extend these findings at the electron microscope level and characterize the spectrum of AP immunoreactivity in neurofibrillary pathology (NFP) of AD and other neurodegenerative disorders including Down's syndrome (DS), Creutzfeldt-Jakob, Parkinson's, Pick's and diffuse Lewy body diseases and progressive supranuclear palsy. In AD and DS, AP immunoreaction product was evident in all the classical amyloid lesions and NFP in a large sample of all cortical areas examined. The distribution and relative intensity of immunostaining was similar to that of thioflavin S staining in serial sections. In many cases, however, plaques and vessels stained by anti-AP serum were not apparent with thioflavin S. Serial sections immunostained with antiserum to amyloid A, C-reactive protein or to other proteins involved in systemic amyloidoses and the acute phase response showed no evidence of staining in any of the cerebral lesions. Electron microscopy confirmed that AP immunoreactivity was associated with the abnormal filaments characteristic of NFP as well as amyloid fibrils found in plaques and vessels showing congophilic amyloid angiopathy. Plaques of Creutzfeldt-Jakob disease, Pick bodies of Pick's disease, tangles and Lewy bodies in Parkinson's disease and a subpopulation of Lewy bodies in the diffuse Lewy body disease coexistent with AD were also stained. With the exception of vessels in two of the five cases, AP was not detected in age-matched controls. Our observations indicate AP to be a consistent feature of cerebral NFP and amyloid deposits.     

Analysis of presenilin 1 and presenilin 2 expression and processing by newly developed monoclonal antibodies.

Because distinct mutations in presenilin 1 and presenilin 2 are a major cause of early-onset familial Alzheimer's disease, we generated four monoclonal antibodies for the identification, localization, and investigation of presenilins in various cell lines and tissues from patients and controls. We show that these antibodies are specific for the N- and C-terminal domains of human presenilin 1 and presenilin 2. They recognize presenilin full-length proteins and their approximately 28-35 kDa N-terminal fragments and approximately 18-20 kDa C-terminal fragments. None of the antibodies showed cross-reaction in their specific detection ability. We demonstrated that presenilin 1 and presenilin 2 are proteolytically processed in human glioma cell lines, transfected and untransfected human neuroblastoma SH-SY5Y cells, COS-7 cells, rat cerebellar neuronal ST15 cells, mouse and human brain. Remarkably, we observed that presenilin 2 is alternatively cleaved during apoptosis, producing smaller C-terminal fragments. By analyzing the subcellular distribution of presenilins, we found reticular and fine vesicular staining throughout the cell bodies. In addition, staining of Golgi compartments and the perinuclear envelope was observed. Alzheimer's disease brain showed strong immunoreactivity of presenilin 1 in reactive astrocytes and senile plaques. This high expression of presenilin 1 may explain the increased production and accumulation of the amyloid-beta peptide in patients with sporadic Alzheimer's disease in the absence of familial presenilin mutation.     

Selective PrP-like protein, doppel immunoreactivity in dystrophic neurites of senile plaques in Alzheimer's disease

Doppel (Dpl) is a prion-like protein encoded by the gene PRND, which has been found downstream of the prion gene PRNP in several species. The present study examines by immunohistochemistry Dpl expression in brain samples from 10 patients with Alzheimer's disease (AD), three patients with Pick's disease, four patients with Parkinson's disease, eight patients with diffuse Lewy body disease (DLBD), six patients with sporadic Creutzfeldt-Jakob disease (CJD) methionine/methionine at the codon 129, two patients with sporadic CJD methionine/valine at the codon 129 and numerous kuru plaques in the cerebellum, one patient with fatal familial insomnia (FFI), and 10 age-matched controls. In the adult human brain, Dpl immunoreactivity was restricted to scattered granule cells of the cerebellum and scattered small granules in the cerebral cortex. Dpl immunoreactivity was seen around betaA4 amyloid deposits in neuritic plaques, but not in diffuse plaques, AD and the common form of DLBD. Neurofibrillary tangles, Pick bodies and Lewy bodies were not stained with anti-Dpl antibodies. No modifications in Dpl immunoreactivity were observed in CJD excepting those associated with accompanying senile plaques. No Dpl-positive deposits were seen in FFI. Whether Dpl in neuritic plaques may attenuate amyloid-induced oxidative stress and participate in the glial response around amyloid cores is discussed in light of the few available data on Dpl functions.     

Cerebral degeneration in Down's syndrome]

Clinical and neuropathological findings of a 63-year-old male and a 25-year-old female with Down's syndrome are presented. Neuropathological examination of the older patient revealed intense features of Alzheimer's disease or senile dementia, including congophilic angiopathy and extensive mineral deposits in the globus pallidus and in the white matter of the cerebellum. In the hippocampus of the younger patient, plaque-like bodies and a few neurofibrillary tangles were found. From a survey of the cases hitherto reported in the literature it appears that among patients over 50 years of age it is common to encounter pathological features typical of Alzheimer's disease or senile dementia, that plaque-like bodies may occur in the second decade, neurofibrillary tangles in the third decade and a congophilic angiopathy in the fourth decade. The congophilic angiopathy is a frequent finding. Due to their high frequency, calcium or calciumlike deposits are regarded as important histopathological substrates of Down's syndrome. The clinical symptomatology of the long-surviving patients with Down's syndrome is that of a non-characteristic brain aging process and differs from that of the typical Alzheimer's disease. Organic dementia is not regularly found. Altogether, the anatomical findings in adult patients with Down's syndrome indicate a premature aging of the brain, which becomes more significant and widespread with increasing age.     

Cerebral amyloid angiopathy: pathogenesis and effects on the ageing and Alzheimer brain

Cerebral amyloid angiopathy (CAA) is a feature of ageing and Alzheimer's disease (AD); it is also associated with intracerebral hemorrhage and stroke. Here, the pathogenesis of CAA and its effects on the brain are reviewed and the possible effects of CAA on therapies for Alzheimer's disease are evaluated. Tracer experiments in animals and observations on human brains suggest that peptides such as A beta are eliminated along the peri-arterial interstitial fluid drainage pathways that are effectively the lymphatics of the brain. In CAA, A beta becomes entrapped in drainage pathways in the walls of cerebral arteries, reflecting a failure of elimination of A beta from the ageing brain. One consequence of failure in clearance of A beta is accumulation of soluble and insoluble A beta associated with cognitive decline in AD. Replacement of vascular smooth muscle cells by A beta occurs in severe CAA with weakening of artery walls and increased risk of vessel rupture and intracerebral hemorrhage. Risk factors for CAA include mutations of the amyloid precursor protein (APP) gene and possession of the epsilon 4 allele of apolipoprotein E. There is also evidence that cerebrovascular disease may be a factor in the failure of elimination of A beta along perivascular pathways in sporadic AD; this would link ageing in cerebral arteries with the pathogenesis of Alzheimer's disease. If therapeutic agents, including anti-A beta antibodies, are to be used to eliminate A beta in the treatment of Alzheimer's disease, the effects of CAA on the treatment and the effects of the treatment on the CAA need to be considered.     

A calcium-stimulated serine protease from monkey brain degrades the beta-amyloid precursor protein.

Amyloid deposition, a histopathological feature of Alzheimer's disease brain, may be the underlying cause of this disease. The isolation of enzymes involved in both the normal and aberrant or alternative processing of the beta-amyloid precursor protein may lead to an understanding of how beta-protein, the major component of amyloid deposits, is formed in the brain parenchyma and vasculature of Alzheimer's disease patients and aged humans. As the same kind of deposits is also found in aged primates, the use of primates will undoubtedly help to understand the mechanisms of amyloid deposition, both spatially and temporally. Here we report the partial purification from adult monkey brain of a calcium-activated serine protease that is immunoreactive with antibodies against cathepsin G and is potentially involved in the abnormal degradation of the beta-amyloid precursor protein. Moreover, immunoreactivity with cathepsin G antibodies was localised to astrocytes in both adult and aged monkey cortex, suggesting that our protease may be expressed in astrocytes.     

Fatty aspirin: a new perspective in the prevention of dementia of Alzheimer's type?

Alzheimer's disease (AD) leads to a dramatic decline in cognitive abilities and memory. A more modest disruption of memory often occurs in normal aging and the same circuits that are devastated through degeneration in AD are vulnerable to sub-lethal age-related changes that alter synaptic transmission. There are numerous indications that aberrant plasticity is critically involved in Alzheimer's. Is ageing itself the major risk factor for AD? Is AD an acceleration of normal ageing? We assume that the ability of the brain is to modify its own structural organization and functioning which is liable to become impaired in ageing until it becomes dramatically impaired in Alzheimer's. Moreover, ageing can compromise the conversion of dietary alpha-linolenic acid (ALA) to docosahexaenoic acid (DHA). DHA regulates synaptogenesis and affects the synaptic structure, and synapse density is reduced in ageing. DHA and newly identified DHA-derived messenger, neuroprotecting D1 (NPD1), protect synapses and decrease the number of activated microglia in the hippocampal system. Delaying AD onset by a few years would reduce the number of the cases of dementia in the community. DHA (and NPD1?) and aspirin induce brain-derived neurotrophic factor (BDNF) protein expression and this protein has a crucial role in neuronal survival. The authors--in view of the increased neuroinflammatory reaction frequently observed during normal brain ageing--suggest the long-term use of "fatty aspirin", an association of DHA and/or NPD1 and aspirin (or nitroaspirin), to postpone, or prevent, the structural neurodegeneration of the brain.     

Ubiquitin-binding protein p62 is present in neuronal and glial inclusions in human tauopathies and synucleinopathies.

We examined the immunoreactivity of ubiquitin-binding protein p62 and its association with ubiquitin (Ub), alpha-synuclein, and paired helical filament (PHF)-tau in the affected brain areas of human tauopathies and synucleinopathies. Ubiquitin-binding protein p62 is a widely expressed protein that can bind to Ub noncovalently and is involved in several signalling pathways, making p62 a candidate regulator of Ub-mediated proteolysis. We show that p62 immunoreactivity co-localizes with neuronal and glial Ub-containing inclusions in Alzheimer's disease, Pick's disease, dementia with Lewy bodies, Parkinson's disease, and multiple system atrophy. This is the first demonstration of a common protein component, apart from Ub, that is present in both PHF-tau and alpha-synuclein inclusions. In both tauo- and synucleinopathies, the staining patterns for p62 and Ub were markedly similar, suggesting that a common mechanism which requires interaction of p62 and Ub contributes to the formation of PHF-tau and alpha-synuclein inclusions.     

Neurotensin immunoreactivity in the human cingulate gyrus, hippocampal subiculum and mammillary bodies. Its potential role in memory processing

Neurotensin immunoreactive neurons comprise the majority of large perikarya in the human subiculum and project axons to the alveus, fimbria, fornix and neuropil of the mammillary bodies. These regions are prominently involved in conditions such as Wernicke's and Alzheimer's disease in which memory is impaired. Neurotensin has potential significance as a peptide in a human brain circuit which may serve a role in memory processing.     

Promyelocytic leukemia nuclear bodies provide a scaffold for human polyomavirus JC replication and are disrupted after development of viral inclusions in progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy is a fatal demyelinating disorder due to human polyomavirus JC infection in which there are viral inclusions in enlarged nuclei of infected oligodendrocytes. We report that the pathogenesis of this disease is associated with distinct subnuclear structures known as promyelocytic leukemia nuclear bodies (PML-NBs). Postmortem brain tissues from 5 patients with the disease were examined. Affected cells with enlarged nuclei contained distinct dot-like subnuclear PML-NBs that were immunopositive for PML protein and nuclear body protein Sp100. Major and minor viral capsid proteins and proliferating cell nuclear antigen, an essential component for DNA replication, colocalized with PML-NBs. By in situ hybridization, viral genomic DNA showed dot-like nuclear accumulation, and by electron microscopy, virus-like structures clustered in subnuclear domains, indicating that PML-NBs are the site of viral DNA replication and capsid assembly. Molecules involved in the ubiquitin proteosome pathway (i.e. ubiquitin and small ubiquitin-like modifier 1) did not accumulate in the nuclei with viral inclusions, indicating that cell degeneration may not be dependent on this pathway. When viral progeny production was advanced, PML-NBs were disrupted. These data suggest that: 1) PML-NBs allow for efficient viral propagation by providing scaffolds, 2) disruption of PML-NBs is independent of the ubiquitin-proteasome pathway, and 3) this disruption probably heralds oligodendrocyte degeneration and the resulting demyelination.     

Detection of novel proteins associated with secondary amyloidosis and Alzheimer's disease by monoclonal antibody

We have established a monoclonal antibody (MoAb) AM34 (IgG1) which was prepared by a hybridoma constructed from fusion between murine myeloma cells and murine splenocytes. Crude amyloid proteins which were used as immunogen, were extracted from the kidney of a patient with rheumatoid arthritis by the distilled water method. This antibody strongly reacted with all 8 cases of secondary amyloidosis, but did not react or very weakly reacted with 17 tissue sections of primary or myeloma-associated amyloidosis. Other amyloid tissues did not give any positive reaction. Interestingly, 6 brain tissues of Alzheimer's disease clearly showed positive staining with this antibody, whereas two apparently normal brain tissues exhibited negative staining. Senile plaque cores, neurofibrillary tangles (weakly stained) and cerebrovascular amyloid in Alzheimer's disease were stained. Absorption of the MoAb AM34 with the crude amyloid proteins abolished the immunoreactivity of the MoAb AM34 not only with the kidney tissue section of the secondary amyloidosis, but also with the above mentioned portions of the brain in the case of Alzheimer's disease. Therefore, these immunohistological data suggest that the MoAb AM34 recognizes common epitope which exists in amyloid deposits of both secondary amyloidosis and Alzheimer's disease. An inhibition test on the kidney section showed that the reactivity of MoAb AM34 was not at all inhibited by the pretreatment of the section with 10 times higher concentration of anti-human amyloid A (AA) MoAb KM268 which was prepared against synthetic peptides of AA protein, suggesting that MoAb AM34 might react with amyloid-related protein other than AA protein. In addition, MoAb KM268 did not react with any lesions in Alzheimer's disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemically-reactive zinc in amyloid plaques, angiopathy, and degenerating neurons of Alzheimer's diseased brains

Excess brain zinc has been implicated in Alzheimer's neuropathology. Here we evaluated that hypothesis by searching the brains of Alzheimer's patients for abnormal zinc deposits. Using histochemical methods, we found vivid Zn2+ staining in the amyloid deposits of dense-core (senile) plaques, in the amyloid angiopathy surrounding diseased blood vessels, and in the somata and dendrites of neurons showing the characteristic neurofibrillary tangles (NFT) of Alzheimer's. In contrast, brains from age-matched, non-demented subjects showed only occasional staining for Zn2+ in scattered neurons and possible plaques. A role of abnormal zinc metabolism in Alzheimer's neuropathology is suggested.     

Ischemic rats as a model in the study of the neurobiological role of human beta-amyloid peptide. Time-dependent disappearing diffuse amyloid plaques in brain

Brains from patients with Alzheimer's disease contain diffuse and senile amyloid plaques. Using an experimental model, we have addressed the issue whether diffuse plaques of amyloid persist, develop with time, or both, in rats injected with human beta-amyloid-(1-42)-peptide for 3 and 12 mon after brain ischemia. Rats receiving beta-amyloid peptide for 3 months after brain ischemia demonstrated widespread diffuse amyloid plaques in hippocampus and cerebral cortex. Neuronal, glial, ependymal, endothelial and pericyte cell bodies were observed filled with beta-amyloid peptide. No staining was observed in control brains. In the group alive 1 year no deposition of human beta-amyloid peptide was observed, too. Direct evidence that diffuse amyloid plaques can disappear in the brain is thus provided for the first time.     

Corpora amylacea could be an indicator of neurodegeneration

We describe an investigation of corpora amylacea (CA) in the brain tissue of Alzheimer's disease (AD) cases and normal ageing controls, using both light (LM) and electron (EM) microscopic techniques. CA populations were shown by routine histological staining of LR White resin sections with methenamine silver and PAS, and were compared with those shown by immunocytochemistry using antibodies to tau, GFAP, tubulin, ubiquitin, β-amyloid and serum amyloid P component in serial sections. All CA were immunoreactive with anti-tau and all were unreactive with anti-β-amyloid. Most were immunoreactive with anti-serum amyloid P component, although this was often weak in AD. CA from normal ageing brain were immunoreactive for proteins that are associated with the neuronal cytoskeleton and cell injury. CA from AD brain shared some of these but differed from those in normal ageing brain by being in much larger number and more variable in their immunoreactivity. In all CA, X-ray microanalysis illustrated the presence of the metallic elements Ca, Fe and Cu. Aluminium, often associated with AD, was not present, even in CA from AD brain. Phosphorus and sulphur, probably from phosphorylated proteins associated with degenerating cytoskeleton elements, were usually detected. In AD brain, the greater numbers of CA and their variable biochemical and elemental composition, when compared with CA in the normal ageing brain, suggests that they may derive from a number of sources both neuronal and glial as a result of the neurodegenerative disease.     

Nucleus basalis (Ch4) and cortical cholinergic innervation in the human brain: observations based on the distribution of acetylcholinesterase and choline acetyltransferase

The nucleus basalis (NB) of the human brain is a large, complex, and highly differentiated structure. Many of its neurons are magnocellular, hyperchromic, isodendritic, acetylcholinesterase-rich, and choline-acetyltransferase-positive. Concurrent histochemical and immunological staining demonstrated that all choline-acetyltransferase-positive NB neurons in the human brain also contain acetylcholinesterase enzyme activity. Only a small minority of acetylcholinesterase-rich magnocellular cell bodies in the NB failed to show choline acetyltransferase immunoreactivity. Sections that were counterstained for Nissl substance showed that 80-90% of all magnocellular neurons in the NB were choline-acetyltransferase-positive and therefore cholinergic. These characteristics, which are very similar to those of the NB in the monkey brain, justified the designation of these cholinergic neurons in the human brain as the Ch4 (or NB-Ch4) complex. On morphological grounds, the compact parts of the human NB-Ch4 complex were divided into distinct sectors which appeared to show a greater level of differentiation than in the monkey brain. In addition to the compact sectors, interstitial elements of NB Ch4 were embedded within adjacent fiber bundles. The putative cortical projections from NB-Ch4 were identified in the form of acetylcholinesterase-rich fibers. These fibers formed a dense plexus in all cortical regions but also displayed laminar and regional variations. Limbic and paralimbic areas had higher concentrations of these fibers than the immediately adjacent neocortical association areas. Alzheimer's disease was associated with a marked depletion of cortical acetylcholinesterase. Two cases of Alzheimer's disease with relatively selective NB-Ch4 cell loss supported the hypothesis that the corticopetal cholinergic pathways in the human brain may have a topographical organization similar to that in the monkey brain.     

Accumulation of NEDD8 in neuronal and glial inclusions of neurodegenerative disorders

NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) is a ubiquitin-like protein that controls vital biological events through its conjugation to members of the cullin family, which are components of certain ubiquitin E3 ligases. Recent studies have shown that NEDD8 is incorporated into Lewy bodies (LBs) in Parkinson's disease, Mallory bodies in alcoholic liver disease and Rosenthal fibres in astrocytoma. In order to examine whether NEDD8 plays a role in the formation of ubiquitinated inclusions, we performed immunohistochemical staining of brain tissue from patients with various neurodegenerative disorders, using an affinity-purified polyclonal antibody raised against NEDD8 that did not cross-react with ubiquitin. In LB disease, NEDD8 immunoreactivity was present in almost all of the LBs and Lewy neurites. Moreover, NEDD8 immunoreactivity was found in a variety of ubiquitinated inclusions, including neuronal and oligodendroglial inclusions in multiple system atrophy, neurofibrillary tangles in Alzheimer's disease, ubiquitinated inclusions in motor neurone disease, and intranuclear inclusions in triplet repeat diseases. These findings suggest that NEDD8 is involved in the formation of various ubiquitinated inclusions via the ubiquitin-proteasome system.