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Results for “Protein Aggregation, Pathological”

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PubMed2000

Aggregation and fibrillization of the recombinant human prion protein huPrP90-231.

According to the "protein-only" hypothesis, the critical step in the pathogenesis of prion diseases is the conformational transition between the normal (PrP(C)) and pathological (PrP(Sc)) isoforms of prion protein. To gain insight into the mechanism of this transition, we have characterized the biophysical properties of the recombinant protein corresponding to residues 90-231 of the human prion protein (huPrP90-231). Incubation of the protein under acidic conditions (pH 3.6-5) in the presence of 1 M guanidine-HCl resulted in a time-dependent transition from an alpha-helical conformation to a beta-sheet structure and oligomerization of huPrP90-231 into large molecular weight aggregates. No stable monomeric beta-sheet-rich folding intermediate of the protein could be detected in the present experiments. Kinetic analysis of the data indicates that the formation of beta-sheet structure and protein oligomerization likely occur concomitantly. The beta-sheet-rich oligomers were characterized by a markedly increased resistance to proteinase K digestion and a fibrillar morphology (i.e., they had the essential physicochemical properties of PrP(Sc)). Contrary to previous suggestions, the conversion of the recombinant prion protein into a PrP(Sc)-like form could be accomplished under nonreducing conditions, without the need to disrupt the disulfide bond. Experiments in urea indicate that, in addition to acidic pH, another critical factor controlling the transition of huPrP90-231 to an oligomeric beta-sheet structure is the presence of salt.

Chromatography, Gel↗

PubMed2004

Alexander disease: putative mechanisms of an astrocytic encephalopathy.

Alexander disease (AXD) is the first primary astrocytic disorder. This encephalopathy is caused by dominant mutations in the glial fibrillary acidic protein (GFAP) gene, encoding the main intermediate filament of astrocyte. Pathologically, this neurodegenerative disease is characterised by dystrophic astrocytes containing intermediate filament aggregates associated with myelin abnormalities. More than 20 GFAP mutations have been reported. Many of them cluster in highly conserved regions between several intermediate filaments. Contrary to other intermediate filament-related diseases, AXD seems to be the consequence of a toxic gain of function induced by aggregates. This is supported by the phenotype of mice overexpressing human GFAP. Nevertheless, GFAP null mice display myelin abnormalities and blood-brain barrier dysfunction that are present in AXD. Given the pivotal role of astrocytes in brain physiology, there are many possibilities for astrocytes to dysfunction and to impair the functions of other cells. Physiopathological hypotheses are discussed in the frame of AXD.

Alexander Disease↗

PubMed1987

[Effect of cryoapheresis on the course of immune complex diseases].

The paper is concerned with an analysis of the results of the method of cryoheparinoprecipitation of plasma proteins in patients with immune complex pathology. The essence of the method is that in plasma cooling cryofibrinogens, cryoglobulins, immune complexes and other components of the complement system aggregate and precipitate. The process is potentiated by adding heparin to the plasma. After removal of cryoproteins the plasma is frozen and returned to a patient during a session of plasmapheresis. The method makes it possible to give up the use of various protein preparations as substitutes. It was applied to 90 patients: 52 females and 38 males aged 15 to 64. The total number of plasmapheresis sessions was 831, an average of 6 sessions per patient; repeated sessions were performed in 20 patients in different time-intervals. Indications for therapy were the absence of a response to routine drug therapeutic methods for a long period of time and steroid-related complications. Cryoapheresis was applied to 18 patients with hemorrhagic vasculitis. Signs of the abdominal, dermal and articular syndrome were eliminated in all the patients; the urinary syndrome was retained in half of the cases necessitating other therapeutic modalities. A CIC level investigated by a method of precipitation with 3.5% polyethylene glycol was not a criterion of assessment of the gravity of diseases: in cases of its increase before therapy initiation there was a clear tendency to a decrease and normalization by the end of treatment. In hemostasis investigation hypercoagulation activation and hyperfibrinogenemia were revealed in most of the patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed2002

Trinucleotide repeat disease. The androgen receptor in spinal and bulbar muscular atrophy.

It has been more than 10 years since the discovery that the expansion of a simple CAG trinucleotide repeat within the coding region of the androgen receptor gene leads to the motor neuronopathy spinal and bulbar muscular atrophy (SBMA). A flurry of investigation into this and the other, more recently discovered, polyglutamine diseases has led to an understanding of many aspects of the molecular pathogenesis of this family of diseases. A characteristics pathological feature of the polyglutamine diseases is the occurrence in affected neurons of ubiquitinated aggregates; such aggregates also contain, among others, proteins involved in the folding and degradation of the mutant proteins. Aggregates themselves are likely not directly cytotoxic, but rather mark the accumulation of all or part of the mutant protein. Furthermore, aggregation occurs because of the inefficient clearance of the mutant protein by the ubiquitin-proteasome pathway for protein degradation. These findings are common to the polyglutamine diseases and reflect the general problem of folding/degrading expanded polyglutamines. In SBMA, the altered metabolism of the androgen receptor is ligand dependent. How the accumulation of the mutant protein causes neuronal dysfunction and disease is not well understood, but several cellular processes have been implicated. Although these findings provide insight into the toxic function of the expanded polyglutamine protein, additional investigations have led to the finding that intrinsic AR transactivational function is somewhat diminished in the presence of the expanded polyglutamine; this likely leads to the partial androgen insensitivity that characterizes patients with SBMA. The recent development of useful animal and cell models of SBMA will lead to increased understanding of disease pathogenesis, as well as to the development of new and better therapeutic strategies.

Animals↗

PubMed2002

Pathogenic theories and intrathecal analysis of the sporadic form of Alzheimer's disease.

Alzheimer's disease (AD) is an age-dependent dementia characterized by progressive loss of cognitive functions and by characteristic pathological changes in the brain: the formation of aggregates extracellularly by beta-amyloid (Abeta) peptide and intracellularly by tau proteins. The disease presents several major diagnostic difficulties: (1) AD develops slowly; (2) analysis of damaged brain tissues is difficult, requiring a biopsy which poses ethical problems; (3) no biochemical markers are available for the diagnosis and monitoring of the disease progression. Since the cerebrospinal fluid (CSF) is in contact with the extracellular space of the brain, many studies have tried to correlate the levels of the intrathecal peptides and amino acids and the development of dementia. The present review analyzes the main results of intrathecal content analyses in light of pathogenic theories proposed to explain the damage associated with AD and observed in the brain of patients by postmortem examination.

Alzheimer Disease↗

PubMed2006

Inhibition of GSK3 dependent tau phosphorylation by metals.

One of the main pathological characteristics of Alzheimer's disease is the presence in the brain of the patients of an aberrant structure, the paired helical filaments, composed of hyperphosphorylated tau. The level of tau phosphorylation has been correlated with the capacity for tau aggregation. Thus, the mechanism for tau phosphorylation could be important to clarify those pathological features in Alzheimer's disease. Tau protein could be modified by different kinases, being GSK3 the one that could modify more sites of that protein. GSK3 activity could be modulate by the presence of metals like magnesium that can be required for the proper function of the kinase, whereas, metals like manganesum or lithium inhibit the activity of the kinase. Many works have been done to study the inhibition of GSK3 by lithium, a specific inhibitor of that kinase. More recently, it has been indicated that sodium tungstate could also inhibit GSK3 through a different mechanism. In this review, we discuss the effect of these two metals, lithium and tungstate, on GSK3 (or tau I kinase) activity.

Animals↗

PubMed2005

Severe muscle disease-causing desmin mutations interfere with in vitro filament assembly at distinct stages.

Desmin is the major intermediate filament (IF) protein of muscle. Recently, mutations of the desmin gene have been reported to cause familial or sporadic forms of human skeletal, as well as cardiac, myopathy, termed desmin-related myopathy (DRM). The impact of any of these mutations on filament assembly and integration into the cytoskeletal network of myocytes is currently not understood, despite the fact that all cause the same histopathological defect, i.e., desmin aggregation. To gain more insight into the molecular basis of this process, we investigated how mutations within the alpha-helical rod domain of desmin affect both the assembly of the recombinant protein in vitro as well as the filament-forming capacity in cDNA-transfected cells. Whereas 6 of 14 mutants assemble into seemingly normal IFs in the test tube, the other mutants interfere with the assembly process at distinct stages, i.e., tetramer formation, unit-length filament (ULF) formation, filament elongation, and IF maturation. Correspondingly, the mutants with in vitro assembly defects yield dot-like aggregates in transfected cells, whereas the mutants that form IFs constitute a seemingly normal IF cytoskeleton in the cellular context. At present, it is entirely unclear why the latter mutant proteins also lead to aggregate formation in myocytes. Hence, these findings may be a starting point to dissect the contribution of the individual subdomains for desmin pathology and, eventually, the development of therapeutic interventions.

3T3 Cells↗

PubMed2003

Energetic characteristics of the new transthyretin variant A25T may explain its atypical central nervous system pathology.

Transthyretin (TTR) is a tetrameric protein that must misfold to form amyloid fibrils. Misfolding includes rate-limiting tetramer dissociation, followed by fast tertiary structural changes that enable aggregation. Amyloidogenesis of wild-type (WT) TTR causes a late-onset cardiac disease called senile systemic amyloidosis. The aggregation of one of > 80 TTR variants leads to familial amyloidosis encompassing a collection of disorders characterized by peripheral neuropathy and/or cardiomyopathy. Prominent central nervous system (CNS) impairment is rare in TTR amyloidosis. Herein, we identify a new A25T TTR variant in a Japanese patient who presented with CNS amyloidosis at age 42 and peripheral neuropathy at age 44. The A25T variant is the most destabilized and fastest dissociating TTR tetramer published to date, yet, surprising, disease onset is in the fifth decade. Quantification of A25T TTR in the serum of this heterozygote reveals low levels relative to WT, suggesting that protein concentration influences disease phenotype. Another recently characterized TTR CNS variant (D18G TTR) exhibits strictly analogous characteristics, suggesting that instability coupled with low serum concentrations is the signature of CNS pathology and protects against early-onset systemic amyloidosis. The low A25T serum concentration may be explained either by impaired secretion from the liver or by increased clearance, both scenarios consistent with A25T's low kinetic and thermodynamic stability. Liver transplantation is the only known treatment for familial amyloid polyneuropathy. This is a form of gene therapy that removes the variant protein from serum preventing systemic amyloidosis. Unfortunately, the choroid plexus would have to be resected to remove A25T from the CSF-the source of the CNS TTR amyloid. Herein we demonstrate that small-molecule tetramer stabilizers represent an attractive therapeutic strategy to inhibit A25T misfolding and CNS amyloidosis. Specifically, 2-[(3,5-dichlorophenyl)amino]benzoic acid is an excellent inhibitor of A25T TTR amyloidosis in vitro.

Amyloid Neuropathies, Familial↗

PubMed1997

[The spectrum of prion pathology broadens: fatal familial insomnia].

INTRODUCTION: The small group of prion diseases, caused by accumulation in the brain of an abnormal protein characterized by its aggregation and relative resistance to proteases (the PrPSc) in man is comprised of Creutzfeldt-Jacob disease (CJE), the Gerstmann-Straussler-Scheinker syndrome, kuru and the newest addition which is fatal familial insomnia (FFI). DEVELOPMENT: FFI is a hereditary condition with dominant autosomal transmission, characterized clinically by progressive insomnia, dysautonomy, changes in the circadian rhythm of hormone secretion, motor signs and slight to moderate deterioration of cognition. The usual age of onset is between 40 and 60 years, and the course of the illness lasts between 7 and 18 months. The histopathological changes, involving neurone loss and reactive gliosis, particularly affect the anteroventral and dorsomedial thalamic nuclei. These lesions lead to insomnia and to autonomic and endocrine disorders. To a lesser extent and degree, lesions are seen in other thalamic nuclei, the cerebral cortex, inferior olives and the cerebellum. FFI and some families with CJE have the same mutation of the codon 178 of the protein prion gene (gene PRNP) with substitution of aspartic acid by asparagine. Polymorphism of codon 129, which codifies methionine or valine determines the development of the clinical and neuropathological phenotype of FFI or CJE respectively. CONCLUSIONS: The description of FFI and the detection of PrPSe in familial cases of diffuse subcortical gliosis has indicated the possibility that there may be other familial or non-familial neurodegenerative diseases caused by prions.

Adult↗

PubMed2003

Specific inhibition of pathological prion protein accumulation by small interfering RNAs.

Development of transmissible spongiform encephalopathies (TSEs) pathogenesis requires the presence of both the normal host prion protein (PrP-sen) and the abnormal pathological proteinase-K resistant isoform (PrP-res). PrP-res forms highly insoluble aggregates, with self-perpetuating properties, by binding and converting PrP-sen molecules into a likeness of themselves. In the present report, we show that small interfering RNA (siRNA) duplexes trigger specific Prnp gene silencing in scrapie-infected neuroblastoma cells. A non-passaged, scrapie-infected culture transfected with siRNA duplexes is depleted of PrP-sen and rapidly loses its PrP-res content. The use of different murine-adapted scrapie strains and host cells did not influence the siRNA-induced gene silencing efficiency. More than 80% of transfected cells were positive for the presence of fluorescein-labeled siRNA duplexes. No cytotoxicity associated with the use of siRNA was observed during the time course of these experiments. Despite a transient abrogation of PrP-res accumulation, our results suggest that the use of siRNA may provide a new and promising therapeutic approach against prion diseases.

Animals↗

PubMed1996

Staging the pathological assembly of truncated tau protein into paired helical filaments in Alzheimer's disease.

Tau protein, which is incorporated into the core of paired helical filaments (PHFs) in Alzheimer's disease (AD), can be characterised immunochemically by C-terminal truncation at Glu-391 recognised by monoclonal antibody (mAb) 423, and acid-reversible occlusion of a generic tau epitope in the tandem repeat region recognised by mAb 7.51. PHFs are also characterised by the presence of binding sites for a fluorescent dye (thiazin red) which can be used to differentiate between amorphous and fibrillar states of tau and beta-amyloid proteins in AD. We have used double labelling confocal microscopy to investigate that state of aggregation of the tau antigens associated with the core structure of the PHF at early stages of neurofibrillary pathology. We report that the early abnormal tau deposits in cells vulnerable to neurofibrillary degeneration are characterised by C-terminal truncation at Glu-391, acid-reversible occlusion of the mAb 7.51 epitope, and the absence of binding sites for thiazin red, consistent with the amorphous non-fibrillar structure demonstrated by immunoelectron microscopy. Transition to the fibrillar state in the PHF is associated with acid-reversible occlusion of both mAb 7.51 and 423 epitopes, and acquisition of binding sites for thiazin red. In neurites, the transition between the two states of aggregation shows distal to proximal polarity, with the fibrillar state found nearest the cell body. These findings demonstrate that the assembly of tau protein into the PHF occurs in at least two stages, an amorphous stage characterised by C-terminal truncation and occlusion of sites within the tandem repeat region, and a fibrillar stage characterised by acid-reversible occlusion of both epitopes via addition of intact tau molecules in the fuzzy coat of the PHF.

Aged↗

PubMed2004

Polyglutamine expansion in ataxin-3 does not affect protein stability: implications for misfolding and disease.

Polyglutamine proteins that cause neurodegenerative disease are known to form proteinaceous aggregates, such as nuclear inclusions, in the neurons of affected patients. Although polyglutamine proteins have been shown to form fibrillar aggregates in a variety of contexts, the mechanisms underlying the aberrant conformational changes and aggregation are still not well understood. In this study, we have investigated the hypothesis that polyglutamine expansion in the protein ataxin-3 destabilizes the native protein, leading to the accumulation of a partially unfolded, aggregation-prone intermediate. To examine the relationship between polyglutamine length and native state stability, we produced and analyzed three ataxin-3 variants containing 15, 28, and 50 residues in their respective glutamine tracts. At pH 7.4 and 37 degrees C, Atax3(Q50), which lies within the pathological range, formed fibrils significantly faster than the other proteins. Somewhat surprisingly, we observed no difference in the acid-induced equilibrium and kinetic un/folding transitions of all three proteins, which indicates that the stability of the native conformation was not affected by polyglutamine tract extension. This has led us to reconsider the mechanisms and factors involved in ataxin-3 misfolding, and we have developed a new model for the aggregation process in which the pathways of un/folding and misfolding are distinct and separate. Furthermore, given that native state stability is unaffected by polyglutamine length, we consider the possible role and influence of other factors in the fibrillization of ataxin-3.

Ataxin-3↗

PubMed2001

Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation.

The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin-proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14-3-3, and alpha-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin-proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.

14-3-3 Proteins↗

PubMed2005

Trehalose reduces aggregate formation and delays pathology in a transgenic mouse model of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease that presents in the fifth or sixth decade with dysphagia, ptosis and proximal limb weakness. OPMD is caused by the abnormal expansion of a polyalanine tract within the coding region of polyA binding protein nuclear 1 (PABPN1). The resultant mutant PABPN1 forms aggregates within the nuclei of skeletal muscle fibres. We have previously described a transgenic mouse model of OPMD that recapitulates the human disease and develops progressive muscle weakness accompanied by the formation of aggregates in skeletal muscle nuclei. The chemical chaperone trehalose has been used effectively to alleviate symptoms in a mouse model of Huntington's disease and is thought to elicit its effect by binding and stabilizing partially folded polyglutamine proteins and inhibiting the formation of aggregates. Here, we show that trehalose reduces aggregate formation and toxicity of mutant PABPN1 in cell models. Furthermore, oral administration of trehalose attenuated muscle weakness, reduced aggregate formation and decreased the number of TUNEL-labelled nuclei in skeletal muscle in an OPMD transgenic mouse model. Thus, anti-aggregation therapy may prove effective in the treatment of human OPMD.

Analysis of Variance↗

PubMedDate not supplied

[Molecular basis of Huntington's disease and possible pathogenic mechanisms].

INTRODUCTION: Huntington s disease is one of the, at least, nine neurological disorders caused by a CAG triplet expansion coding for a poly glutamine sequence in the corresponding protein. Huntington s disease affects 3 7 in 100.000 individuals in Western Europe descendent population and the symptomatology comprises motor (including chorea and rigidity), cognitive (subcortical dementia), and psychological (including irritability and depression) manifestations until death. DEVELOPMENT: Neuropathology is extremely restricted, with atrophy occurring in the striatum and, to a lesser extent, in the cerebral cortex. Microscopically, the neuropathology is characterized by neuronal loss, reactive gliosis, and intraneuronal protein aggregates. Since the initial description of this disease by George Huntington in 1872, substantial advance has been achieved in the understanding of this pathology. The pathogenic gene and mutation were identified in 1993. This allowed the generation of multiple in vitro, cellular, and animal models of Huntington s disease. These studies have originated multiple hypotheses regarding the mechanism by which huntingtin with an expanded poly glutamine tract exerts its toxicity. CONCLUSION: We try to summarize the current knowledge about this disease from the clinical manifestations to the molecular basis, in an attempt to offer a global view of this pathology.

Humans↗

PubMedDate not supplied

The early cellular pathology of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder that affects about one in 10,000 individuals in North America. The genetic defect responsible for the disease is an expansion of a CAG repeat that encodes a polyglutamine tract in the expressed protein, huntingtin. The disease is characterized by involuntary movements, cognitive impairment, and emotional disturbance. Despite the widespread expression of huntingtin, the brains of HD patients show selective neuronal loss in the striatum and the deep layers of the cerebral cortex. Recent studies have shown that polyglutamine expansion causes huntingtin to aggregate, to accumulate in the nucleus, and to interact abnormally with other proteins. Several cellular and animal models for HD have revealed that intranuclear accumulation of mutant huntingtin and the formation of neuropil aggregates precede neurological symptoms and neurodegeneration. Intranuclear huntingtin may affect nuclear function and the expression of genes important for neuronal function, whereas neuropil aggregates may interfere with neuritic transport and function. These early pathological events, which occur in the absence of neurodegeneration, may contribute to the neurological symptoms of HD and ultimately lead to neuronal cell death.

Animals↗

PubMedDate not supplied

A human single-chain Fv intrabody preferentially targets amino-terminal Huntingtin's fragments in striatal models of Huntington's disease.

Amino-terminal fragments of huntingtin (htt) appear to result from proteolytic processing of the full-length protein in Huntington's disease (HD), and fragments containing pathological expansions of polyglutamine elicit toxicity in model systems. Such fragments are sequestered into insoluble aggregates, which may initially serve a cellular protective mechanism, while soluble fragments and/or oligomers may be a more acute toxic species. Agents which enhance mutant htt clearance have shown therapeutic potential in animal models of HD. Here, we present the first evidence of an htt-specific single-chain Fv intrabody (C4) that selectively targets the soluble fraction of amino-terminal htt fragments. Our findings suggest that the C4 intrabody binds weakly, but does not alter the levels of endogenous, full-length htt. C4 appears to decrease the steady-state levels of amino-terminal htt fragments by binding to non-aggregated, but not aggregated, htt species. Intrabodies may be used as potential curative agents, and as drug discovery tools, for HD and other misfolded protein disorders.

Animals↗

PubMed2005

A precipitating role for truncated alpha-synuclein and the proteasome in alpha-synuclein aggregation: implications for pathogenesis of Parkinson disease.

Parkinson disease and other alpha-synucleinopathies are characterized by the deposition of intraneuronal alpha-synuclein (alphaSyn) inclusions. A significant fraction (about 15%) of alphaSyn in these pathological structures are truncated forms that have a much higher propensity than the full-length alphaSyn to form aggregates in vitro. However, little is known about the role of truncated alphaSyn species in pathogenesis or the means by which they are generated. Here, we have provided an in vitro mechanistic study demonstrating that truncated alphaSyns induce rapid aggregation of full-length protein at substoichiometric ratios. Co-overexpression of truncated alphaSyn with full-length protein increases cell vulnerability to oxidative stress in dopaminergic SH-SY5Y cells. These results suggest a precipitating role for truncated alphaSyn in the pathogenesis of diseases involving alphaSyn aggregation. In this regard, the A53T mutation found in some cases of familial Parkinson disease exacerbates the accumulation of insoluble alphaSyns that correlates with the onset of pathology in transgenic mice expressing human alphaSyn-A53T mutant. The caspase-like activity of the 20 S proteasome produces truncated fragments similar to those found in patients and animal models from degradation of unstructured alphaSyn. We propose a model in which incomplete degradation of alphaSyn, especially under overloaded proteasome capacity, produces highly amyloidogenic fragments that rapidly induce the aggregation of full-length protein. These aggregates in turn reduce proteasome activity, leading to further accumulation of fragmented and full-length alphaSyns, creating a vicious cycle of cytotoxicity. This model has parallels in other neurodegenerative diseases, such as Huntington disease, where coaggregation of poly(Q) fragments with full-length protein has been observed.

Aged↗