Accumulation Of Altered Proteins Research Articles

α-Synuclein Aggregates with β-Amyloid or Tau in Human Red Blood Cells: Correlation with Antioxidant Capability and Physical Exercise in Human Healthy Subjects.

Neurodegenerative disorders (NDs) are characterized by abnormal accumulation/misfolding of specific proteins, primarily α-synuclein (α-syn), β-amyloid1-42 (Aβ), and tau, in both brain and peripheral tissue. In addition to homo-oligomers, the role of α-syn interactions with Aβ or tau has gradually emerged. The altered protein accumulation has been related to both oxidative stress and physical activity; nevertheless, no correlation among the presence of peripheral α-syn hetero-aggregates, antioxidant capacity, and physical exercise has been discovered as of yet. Herein, the content of α-syn, Aβ, tau, and of their heterocomplexes was determined in red blood cells (RBCs) of healthy subjects (sedentary and athletes). Such parameters were related to the extent of the antioxidant capability (AOC), a key marker of oxidative stress in aging-related pathologies, and to physical exercise, which is known to play an important preventive role in NDs and to modulate oxidative stress. Tau content and plasma AOC toward hydroxyl radicals were both reduced in older or sedentary subjects; in contrast, α-syn and Aβ accumulated in elderly subjects and showed an inverse correlation with both hydroxyl AOC and the level of physical activity. For the first time, α-syn heterocomplexes with Aβ or tau were quantified and demonstrated to be inversely related to hydroxyl AOC. Furthermore, α-syn/Aβ aggregates were significantly reduced in athletes and inversely correlated with physical activity level, independent of age. The positive correlation between antioxidant capability/physical activity and reduced protein accumulation was confirmed by these data and suggested that peripheral α-syn heterocomplexes may represent new indicators of ND-related protein misfolding.

Defects in trafficking bridge Parkinson's disease pathology and genetics

Parkinson's disease is a debilitating, age-associated movement disorder. A central aspect of the pathophysiology of Parkinson's disease is the progressive demise of midbrain dopamine neurons and their axonal projections, but the underlying causes of this loss are unclear. Advances in genetics and experimental model systems have illuminated an important role for defects in intracellular transport pathways to lysosomes. The accumulation of altered proteins and damaged mitochondria, particularly at axon terminals, ultimately might overwhelm the capacity of intracellular disposal mechanisms. Cell-extrinsic mechanisms, including inflammation and prion-like spreading, are proposed to have both protective and deleterious functions in Parkinson's disease.

Amyloid generation and dysfunctional immunoproteasome activation with disease progression in animal model of familial Alzheimer's disease.

Double-transgenic amyloid precursor protein/presenilin 1 (APP/PS1) mice express a chimeric mouse/human APP bearing the Swedish mutation (Mo/HuAPP695swe) and a mutant human PS1-dE9 both causative of familial Alzheimer's disease (FAD). Transgenic mice show impaired memory and learning performance from the age of 6 months onwards. Double-transgenic APP/PS1 mice express altered APP and PS1 mRNAs and proteins, reduced β-secretase 1 (BACE1) mRNA and normal BACE1 protein, all of which suggest a particular mechanism of amyloidogenesis when compared with sporadic AD. The first β-amyloid plaques in APP/PS1 mice appear at 3 months, and they increase in number and distribution with disease progression in parallel with increased levels of brain soluble β-amyloid 1-42 and 1-40, but also with reduced 1-42/1-40 ratio with age. Amyloid deposition in plaques is accompanied by altered mitochondria and increased oxidative damage, post-translational modifications and accumulation of altered proteins at the dystrophic neurites surrounding plaques. Degradation pathways are also modified with disease progression including activation of the immunoproteasome together with variable alterations of the different protease activities of the ubiquitin-proteasome system. Present observations show modifications in the production of β-amyloid and activation and malfunction of the subcellular degradation pathways that have general implications in the pathogenesis of AD and more particularly in specificities of FAD amyloidogenesis.

Proteotoxicity and the Contrasting Effects of Oxaloacetate and Glycerol onCaenorhabditis elegansLife Span: A Role for Methylglyoxal?

Because accumulation of altered proteins is the most common biochemical symptom of aging, it is at least possible that such proteotoxicity may cause aging and influence life span. The life span of the nematode worm Caenorhabditis elegans is strongly influenced by changes in the intracellular concentration of methylglyoxal (MG), a putative source of much age-related proteotoxicity and organelle, cellular, and molecular dysfunction. Glycerol has recently been shown to shorten, whereas oxaloacetate has been found to extend, life span in C. elegans. It is suggested here that glycerol and oxaloacetate exert opposing effects on MG formation in C. elegans. It is proposed that, if not secreted by aquaporin, glycerol is converted to glycerol phosphate and then to dihydroxyacetone phosphate (DHAP) via a reaction requiring nicotinamide adenine dinucleotide (NAD(+)). This inhibits operation of the glycerol phosphate cycle in which DHAP is converted into glycerol phosphate, which concomitantly regenerates NAD(+) from NADH, thereby ensuring glycolytic oxidation of glyceraldehyde-3-phosphate (G3P). Because DHAP and G3P spontaneously decompose into MG, and NAD(+) is required for conversion of G3P into phosphoglycerate, the glycerol-induced increased DHAP formation and decreased NAD(+) availability will increase the potential for MG generation. In contrast, oxaloacetate may decrease MG generation by stimulating the operation of the malate-oxaloacetate shuttle, in which oxaloacetate is converted to malate, which regenerates NAD(+) from NADH. By the ensuing G3P oxidation, increased NAD(+) availability will decrease the potential for MG formation. It should be noted that mitochondria are involved in the operation of the above cycle/shuttles and that increased NAD(+) availability also stimulates those sirtuin activities that increase mitogenesis and mitochondrial activity via effects on signal transduction and gene expression, which frequently accompany dietary restriction-induced life span extension.

NAD + and metabolic regulation of age-related proteoxicity: A possible role for methylglyoxal?

A common biochemical characteristic of aging is proteotoxicity i.e. the accumulation of altered proteins. While many studies have shown that NAD + is important when lifespan is modulated by dietary means, it is uncertain whether or how NAD + affects either formation or elimination of altered proteins. It is suggested here that changes in NAD + availability can affect generation of methylglyoxal (MG) from glycolytic intermediates, which in turn can damage proteins, promote generation of reactive oxygen species and induce mitochondrial dysfunction. The proposal can also help to explain how, by altering NAD + regeneration from NADH, dietary supplementation with glycerol or glucose, which decreases lifespan in the nematode Caenorhabditis elegans, could cause MG generation to increase, whereas oxaloacetate supplementation, which increases lifespan, could lower the potential for MG formation. The proposal also suggests how upregulation of mitogenesis and mitochondrial activity, and increased aerobic activity, help to decrease the potential for MG formation and resultant proteotoxicity, with consequential beneficial effects with respect to aging.

Glycation Isotopic Labeling with 13C-Reducing Sugars for Quantitative Analysis of Glycated Proteins in Human Plasma

Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mm glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [(13)C(6)]glucose incubation to evaluate the native glycated proteins labeled with [(12)C(6)]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive approach was also tested to detect potential glycation sites under high glucose concentration.

Error-protein metabolism and ageing

Ageing and many associated pathologies are accompanied by accumulation of altered proteins. It is suggested that erroneous polypeptide biosynthesis, cytosolic and mitochondrial, is not an insignificant source of aberrant protein in growing and non-mitotic cells. It is proposed that (i) synthesis of sufficient proteases and chaperone proteins necessary for rapid elimination of altered proteins, from cytoplasmic and mitochondrial compartments, is related to cellular protein biosynthetic potential, and (ii) cells growing slowly, or not at all, automatically generate lower levels of protease/chaperone molecules than cells growing rapidly, due to decreased general rate of protein synthesis and lowered amount of error-protein produced per cell. Hence the increased vulnerability of mature organisms may be explained, at least in part, by the decline in constitutive protease/chaperone protein biosynthesis. Upregulation of mitochondria biogenesis, induced by dietary restriction or aerobic exercise, may also increase protease/chaperone protein synthesis, which would improve cellular ability to degrade both error-proteins and proteins damaged post-synthetically by reactive oxygen species etc. These proposals may help explain, in part, the latency of those age-related pathologies where altered proteins accumulate only late in life, and the beneficial effects of aerobic exercise and dietary restriction.

P.P.3 04 Autophagic vacuolar myopathies: Immunochemical and molecular characterization

Elucidation of mechanisms involved in toxic protein accumulation and vacuole formation in inclusion body myopathies. Accumulation of altered proteins is a common pathogenic mechanism in several neurodegenerative disorders. Skeletal muscle from sporadic inclusion body myositis (s-IBM) and hereditary inclusion body myopathy (h-IBM) patients, similarly to the nervous system in neurodegenerative disorders, typically contains intracellular aggregated protein depositions such as beta amyloid, tau and prion proteins. We examined muscle biopsies from 34 patients with hIBM, sIBM, oculopharyngeal muscle dystrophy, Danon disease, distal dystrophy with rimmed vacuoles, acid maltase deficiency, and genetically unclassified autophagic vacuolar myopathies with distal or proximal weakness. We studied by immunohistochemistry or immunoblot the expression of components of the proteolytic pathways (lysosomal/endosomal, Ca2+-dependent and ubiquitin-proteasomal), and the expression of toxic proteins and of proteins involved in membrane vesicle fusion and membrane repair. We also analysed the GNE gene in 10 patients. We identified abnormal accumulation of toxic proteins and of ubiquitin proteasomal proteins in most examined muscles. Lysosomal/endosomal components were variably expressed in the different pathologies. Vacuoles were positive for sarcolemmal proteins in most cases and accumulated dysferlin in sIBM and in some genetically unclassified autophagic vacuolar myopathies. GNE mutations were detected in two patients. Histopathological and immunochemical features of the autophagic vacuoles may help to distinguish the different clinical entities and give some hints on the origin of vacuoles.

CYTOCHROME P-450-MEDIATED DIFFERENTIAL OXIDATIVE MODIFICATION OF PROTEINS: ALBUMIN, APOLIPOPROTEIN E, AND CYP2E1 AS TARGETS

Although many studies established a role of cytochrome P-450s in metabolism of xenobiotics, few studies evaluating the ability of cytochrome P-450s to oxidize proteins have been reported. The ability of cytochrome P-450s to induce oxidative modification of albumin, apolipoprotein E, and CYP2E1 protein was investigated. Microsomal cytochrome P-450s induced production of reactive radical species, leading to differential modification of the proteins. Albumin remained unmodified, and CYP2E1 protein was degraded, whereas recombinant and endogenous apolipoprotein E was aggregated. The modification of apolipoprotein E was isoform independent. Cytochrome P-450 inhibitors or antioxidants inhibited the production of reactive radical species and protein modification. These results demonstrate that response of each protein to cytochrome P-450-mediated oxidative attack is different, and cytochrome P-450s can induce apolipoprotein E aggregation, a process that might be relevant to accumulation of altered protein in various abnormal conditions. In view of the ubiquitous expression of cytochrome P-450s, the present results may have important toxicological implications.

Proteomic analysis reveals upregulation of calreticulin in murine dopaminergic neuronal cells after treatment with 6-hydroxydopamine

We have utilized integrated technologies including separation of proteins by 2-dimensional (2-D) gel electrophoresis and identification of proteins by matrix assisted laser desorption/ionizing time of flight (MALDI-TOF) mass spectrometry to examine an array of proteins that are regulated following treatment with neurotoxin. In essence, total cellular lysates harvested from MN9D dopaminergic neuronal cells treated with 6-hydroxydopamine (6-OHDA) for various time periods were subjected to 2-D gel separation followed by an analysis of the protein spots separated. Among the several protein spots that appeared to be either up- or down-regulated following 6-OHDA treatment, MALDI-TOF mass spectrometry revealed that an ER chaperone protein, calreticulin, was upregulated in a time-dependent manner. 6-OHDA-mediated up-regulation of this protein spot was reversed to the untreated control level when MN9D cells were co-treated with a pan-caspase inhibitor or an anti-oxidant. Immunoblot analysis using anti-calreticulin antibody confirmed this phenomenon. Since accumulation of altered proteins may be relevant in Parkinson's disease, our data suggest that regulation of chaperone activity in dopaminergic neurons comprises an additional cellular response to death-inducing stimuli.

Proteomic analysis reveals upregulation of calreticulin in murine dopaminergic neuronal cells after treatment with 6-hydroxydopamine

We have utilized integrated technologies including separation of proteins by 2-dimensional (2-D) gel electrophoresis and identification of proteins by matrix assisted laser desorption/ionizing time of flight (MALDI-TOF) mass spectrometry to examine an array of proteins that are regulated following treatment with neurotoxin. In essence, total cellular lysates harvested from MN9D dopaminergic neuronal cells treated with 6-hydroxydopamine (6-OHDA) for various time periods were subjected to 2-D gel separation followed by an analysis of the protein spots separated. Among the several protein spots that appeared to be either up- or down-regulated following 6-OHDA treatment, MALDI-TOF mass spectrometry revealed that an ER chaperone protein, calreticulin, was upregulated in a time-dependent manner. 6-OHDA-mediated up-regulation of this protein spot was reversed to the untreated control level when MN9D cells were co-treated with a pan-caspase inhibitor or an anti-oxidant. Immunoblot analysis using anti-calreticulin antibody confirmed this phenomenon. Since accumulation of altered proteins may be relevant in Parkinson's disease, our data suggest that regulation of chaperone activity in dopaminergic neurons comprises an additional cellular response to death-inducing stimuli.

Protein misfolding in Alzheimer's and Parkinson's disease: genetics and molecular mechanisms.

The accumulation of altered proteins is a common pathogenic mechanism in several neurodegenerative disorders. A causal role of protein aggregation was originally proposed in Alzheimer's disease (AD) where extracellular deposition of beta-amyloid (Abeta) is the main neuropathological feature. It is now believed that intracellular deposition of aggregated proteins may be relevant in Parkinson's disease (PD), amyotrophic lateral sclerosis and polyglutamine disorders. An impairment of ubiquitin-proteasome system (UPS) appears directly involved in these disorders. We reviewed the results on the role of protein misfolding in AD and PD and the influence of mutations associated with these diseases on the expression of amyloidogenic proteins. Results of genetic screening of familial cases of AD and PD are summarized. In the familial AD population (70 subjects) we found several mutations of the presenilin 1 (PS1) gene with a frequency of 12.8% and one mutation in the gene encoding the protein precursor of amyloid (APP) (1.4%). One mutation of Parkin in the homozygous form and two in the heterozygous form were identified in our PD population. We also reported data obtained with synthetic peptides and other experimental models, for evaluation of the pathogenic role of mutations in terms of protein misfolding.

Aging of proteins and the proteasome.

The progressive and irreversible decline of the different physiological functions of the organism during the last part of its life, a phenomenon commonly referred to as aging, is a complex process that is under some genetic control but is also linked to influences from the environment. Accumulation of damaged protein is a common feature of cellular aging (Berlett and Stadtman 1997; Beckman and Ames 1998). Proteins are indeed the target for different post-translational modifications (oxidation, glycation, glycoxidation, conjugation with lipid peroxidation products) that increase with age (Stadtman 1992). These modifications, which directly alter their biological function, are strongly believed to be implicated in the aging process. The role of several genes is involved in the stress response (e.g., oxidative stress), their overexpression leads to increased longevity (Larsen 1993; Vanfleteren 1993; Orr and Sohal 1994; Hekimi et al. 1998), and the observed age-related increase of damaged cellular components indicates a crucial importance of “maintenance” systems in the aging process. The accumulation of altered proteins with age raises the problem of the efficacy of the proteolytic systems in charge of eliminating these damaged proteins, in particular the efficacy of the proteasomal system, which is implicated not only in the removal of altered proteins, but also in the continuous renewal of intracellular proteins (Coux et al. 1996; Grune et al. 1997; Voges et al. 1999).KeywordsProteasome ActivityProteasomal SystemProteasome SubunitMethionine SulfoxideMulticatalytic ProteinaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Age-related changes in the 20S and 26S proteasome activities in the liver of male F344 rats

Accumulation of altered proteins in old animals has been ascribed to slower turnover of proteins. Since proteasomes can be regarded as the major proteolytic enzymes responsible for the degradation of the majority of cellular proteins, we examined age-related changes of 20S and 26S proteasomes in the liver of young (8–10-month-old), middle-aged (15–18-month-old) and old (25–28-month-old) Fischer 344 male rats. The two forms of proteasomes were separated by glycerol gradient centrifugation. Fluorogenic peptides were used as substrates to evaluate three types of peptidase activities. The ratio of peptidase activities in the 20S proteasome vs. those in the 26S form did not appear to change with age. Unstimulated chymotrypsin-like activity found only in the 26S form decreased by 30% in the old rats as compared with that in the young ones, while no change in the activity was observed during aging when stimulated by sodium dodecyl sulfate. The trypsin-like activity declined significantly by 17% to an apparently similar extent in both 20S and 26S forms. The peptidylglutamyl peptide hydrolyzing activity exhibited gradual decrease with age, resulting in 60% lower value in the old rats as compared with the young animals. These changes are considered to account for the age-related extension of half-life of proteins. Since the amount of total proteasomes measured by immunoblot did not appear to change with age, posttranslational modifications or subunit replacement is possibly responsible for the decrease in the activities.

Deficiency of a protein-repair enzyme results in the accumulation of altered proteins, retardation of growth, and fatal seizures in mice.

L-Asparaginyl and L-aspartyl residues in proteins are subject to spontaneous degradation reactions that generate isomerized and racemized aspartyl derivatives. Proteins containing L-isoaspartyl and D-aspartyl residues can have altered structures and diminished biological activity. These residues are recognized by a highly conserved cytosolic enzyme, the protein L-isoaspartate(D-aspartate) O-methyltransferase (EC 2.1.1.77). The enzymatic methyl esterification of these abnormal residues in vitro can lead to their conversion (i.e., repair) to normal L-aspartyl residues and should therefore prevent the accumulation of potentially dysfunctional proteins in vivo as cells and tissues age. Particularly high levels of the repair methyltransferase are present in the brain, although enyzme activity is present in all vertebrate tissues. To define the physiological relevance of this protein-repair pathway and to determine whether deficient protein repair would cause central nervous system dysfunction, we used gene targeting in mouse embryonic stem cells to generate protein L-isoaspartate(D-aspartate) O-methyltransferase-deficient mice. Analyses of tissues from methyltransferase knockout mice revealed a striking accumulation of protein substrates for this enzyme in the cytosolic fraction of brain, heart, liver, and erythrocytes. The knockout mice showed significant growth retardation and succumbed to fatal seizures at an average of 42 days after birth. These results suggest that the ability of mice to repair L-isoaspartyl- and D-aspartyl-containing proteins is essential for normal growth and for normal central nervous system function.

Changes in superoxide dismutase activity in liver and lung of old rats.

Superoxide dismutase activity was measured in liver and lung from 3 and 24 month-old rats. Both total SOD and Mn-SOD activity decreased significantly in the liver of old rats. Recent results from our laboratory have indicated that during aging, the activity of Cu/Zn-SOD decreases in rat liver and that there is an accumulation of altered protein. It was also shown that the old Cu/Zn-SOD had one histidine fewer than the young one. In the present study, the immunoprecipitation experiments showed that the amount of immunoprecipitable Mn-SOD from liver of old rats was greater than from young ones, but when amino acid residues were measured in purified young and old Mn-SOD from liver, no change was observed. In lung, no significant age-related differences in total SOD, Cu/Zn-SOD and Mn-SOD activity were found, nor was there accumulation of altered protein during aging.

Age related modifications of soluble proteins in various organs of male garden lizard, Calotes versicolor.

The oxidative modification of proteins measured as carbonyl derivatives increased with advancing age in the liver of male garden lizard. The same parameter did not show a significant change in other organs (brain, heart and kidney). Based on the observations in both homeotherms (mammals) and poikilotherms (insect and reptile), the in vivo oxidative modification of cellular proteins appears to be the most common mechanism leading to accumulation of altered proteins during aging. However, the degree of modifications may vary among the tissues/organs of a species. On the other hand the variations may also account for the role of modification of proteins in the pattern of aging in different species.

Oxidative stress and recovery from oxidative stress are associated with altered ubiquitin conjugating and proteolytic activities in bovine lens epithelial cells.

Roles for ubiquitin (an 8.5 kDa polypeptide) involve its conjugation to proteins as a signal to initiate degradation and as a stress protein. We investigated ubiquitin conjugation and ubiquitin-dependent proteolytic activities in cultured bovine lens epithelial cells (BLECs) upon oxidative challenge. A 44% decrease in intracellular glutathione confirmed oxidative stress upon incubation with 1 mM H2O2. After 30 min incubation, endogenous high-molecular-mass ubiquitin conjugates decreased 73%, and intracellular proteolysis decreased about 50%. In the supernatants of the oxidatively treated BLECs, the ability to form high-molecular-mass ubiquitin conjugates with exogenous 125I-labelled ubiquitin decreased 28%, and ATP-dependent degradation of oxidized alpha-crystallin decreased 36%. When the H2O2-treated BLECs were allowed to recover for 60 min, intracellular proteolysis returned to the level of control cells. There was also a subsequent transient enhancement of intracellular proteolysis and a simultaneous recovery of endogenous high-molecular-mass ubiquitin conjugates. In parallel cell-free experiments, conjugating activity with exogenous 125I-labelled ubiquitin and ATP-dependent degradation of oxidized alpha-crystallin increased 35% and 72% respectively compared with non-oxidatively treated BLECs. ATP-independent proteolysis showed little response to exposure or removal of H2O2. These results indicate that (1) the rate of intracellular proteolysis in BLECs is associated with the level of endogenous high-molecular-mass ubiquitin conjugates and (2) oxidative stress may inactivate the ubiquitin conjugation activity with coordinate depression of proteolytic capability. Enhancement in ubiquitin conjugation and proteolytic activities during recovery from oxidative stress may be important in removal of damaged proteins and restoration of normal function of BLECs. The inactivation of ubiquitin-dependent proteolysis by oxidation may be involved in the accumulation of altered proteins and other adverse sequelae in the oxidatively challenged aging lens.

Immunochemical identification of ubiquitin and heat-shock proteins in corpora amylacea from normal aged and Alzheimer's disease brains.

Corpora amylacea (CA) accumulation in the central nervous system (CNS) is associated with both normal aging and neurodegenerative conditions such as Alzheimer's disease (AD). CA is reported to be primarily composed of glucose polymers, but approximately 4% of the total weight of CA is consistently composed of protein. CA protein resolved on sodium dodecylsulfate-polyacrylamide gel electrophoresis showed a broad range of polypeptides ranging from 24 to 133 kDa, with four abundant bands. Immunoblots of the profile of polypeptides solubilized from purified CA, showed positive ubiquitin (Ub) immunoreactivity for all the bands. Antisera to heat-shock proteins (hsp) 28 and 70 reacted selectively with bands of 30 and 67 kDa. These results show that Ub is associated with the primary protein components of CA and that the polypeptides are likely to be Ub conjugates. Immunostaining experiments were performed to specifically characterize the protein components of CA in brain tissue sections as well as those of CA purified from both AD and normal aged brains. In all cases CA showed positive reactions with antibodies to Ub, with antibodies raised against either paired helical filaments or hsp 28 or 70, the most prominent staining being with antibodies to Ub, hsp 28 or hsp 70. The presence of Ub and hsp 28 and 70, which are actively induced after stress, suggests that accumulation of altered proteins, possibly attributed to an increased frequency of unusual post-translational modifications or to a sustained physiological stress (related to both normal aging and neurodegenerative process), may be involved in the pathogenesis of CA.