Encephalopathy Prions Research Articles

Insulin-like Growth Factor-I Prevents the Accumulation of Autophagic Vesicles and Cell Death in Purkinje Neurons by Increasing the Rate of Autophagosome-to-lysosome Fusion and Degradation

Continuous macroautophagic activity is critical for the maintenance of neuronal homeostasis; however, unchecked or dysregulated autophagy can lead to cell death. Cultured Purkinje neurons die by an autophagy-associated cell death mechanism when deprived of trophic support. Here, we report that insulin-like growth factor-I (IGF-I) completely blocked the autophagy-associated cell death of Purkinje neurons. To examine the mechanism by which IGF-I influences autophagy, neurons were infected with adeno-RFP-LC3 and subjected to trophic factor withdrawal, and the size and number of autophagosomes were analyzed by live-cell fluorescence imaging. In control neurons, autophagy occurred at a constitutive low level with most autophagosomes measuring less than 0.75 microm. Trophic factor withdrawal increased the number and size of autophagosomes with most autophagosomes ranging between 0.75 and 1.5 microm and some reaching 1.5-2.25 microm. IGF-I added at the time of trophic factor withdrawal prevented the accumulation of the larger autophagosomes; however, it had no effect on the conversion of LC3, an indicator of autophagy induction. Instead, the rate of autophagosome-to-lysosome fusion measured by colocalization of RFP-LC3 and LysoSensor Green was accelerated by IGF-I. Treating the neurons with bafilomycin A(1) in the presence of IGF-I led to the accumulation of autophagosomes even larger than those induced by trophic factor withdrawal alone, indicating that IGF-I regulates autophagic vesicle turnover. Finally, the effect of IGF-I on autophagy was mediated by an Akt/mTOR-de pend ent and an ERK-independent pathway. These data suggest a novel role for IGF-I in protecting Purkinje neurons from autophagy-associated cell death by increasing autophagy efficiency downstream of autophagy induction.

Peptide Fragment Approach to Prion Misfolding: The Alpha-2 Domain

The mechanism that underlies a multitude of human disorders, including type II diabetes, Parkinson’s, Huntington’s and Alzheimer’s, and the prion encephalopathies, is β-structure expansion through a pathogenic aggregation-prone monomeric form. β-sheet expansion disorders share intermolecular association as a common determinant, being therefore collectively identified as conformational diseases, but little is known about the underlying mechanism. Transmissible spongiform encephalopathies, also known as prion diseases, are all characterised by progressive neuronal degeneration associated to marked extracellular accumulation of an amyloidogenic conformer of the normal cellular prion protein (PrPC), referred to as the scrapie isoform (PrPSc), which is thought to be responsible for the disease symptoms. PrPC is a ubiquitous 231-amino acid glycoprotein, whose physiological role is still elusive. It is organised as an N-terminal disordered region and a compact C-terminal domain, where secondary structure elements consist of three α-helices (α1, α2 and α3), with an α2-α3 disulphide bridge, and two short β-strands (β1 and β2). Evidence accumulated so far suggests that the protein possesses one or several ‘spots’ of intrinsic conformational weakness, which may trigger generic folding, leading the whole architecture to adopt aggregation-prone conformations. One of such spots is suspected to be the C-terminal side of the α-helix 2, which has recently gained the attention of several investigations because it gathers several disease-associated point mutations, can be strongly fibrillogenic and toxic to neuronal cells, and possesses chameleon conformational behaviour. This paper briefly reviews recent literature on α-2 domain-derived model peptides.

Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization

Repeating intermolecular protein association by means of beta-sheet expansion is the mechanism underlying a multitude of diseases including Alzheimer's, Huntington's and Parkinson's and the prion encephalopathies. A family of proteins, known as the serpins, also forms large stable multimers by ordered beta-sheet linkages leading to intracellular accretion and disease. These 'serpinopathies' include early-onset dementia caused by mutations in neuroserpin, liver cirrhosis and emphysema caused by mutations in alpha(1)-antitrypsin (alpha(1)AT), and thrombosis caused by mutations in antithrombin. Serpin structure and function are quite well understood, and the family has therefore become a model system for understanding the beta-sheet expansion disorders collectively known as the conformational diseases. To develop strategies to prevent and reverse these disorders, it is necessary to determine the structural basis of the intermolecular linkage and of the pathogenic monomeric state. Here we report the crystallographic structure of a stable serpin dimer which reveals a domain swap of more than 50 residues, including two long antiparallel beta-strands inserting in the centre of the principal beta-sheet of the neighbouring monomer. This structure explains the extreme stability of serpin polymers, the molecular basis of their rapid propagation, and provides critical new insights into the structural changes which initiate irreversible beta-sheet expansion.

Serpins show structural basis for oligomer toxicity and amyloid ubiquity

Many disorders, including Alzheimer’s, the prion encephalopathies and other neurodegenerative diseases, result from aberrant protein aggregation. Surprisingly, cellular toxicity is often due not to the highly-ordered aggregates but to the oligomers that precede their formation. Using serpins as a paradigm, we show how the active and infective interface of oligomers is inherently toxic and can promiscuously bind to unrelated peptides, including neurotransmitters. Extension of the oligomer and its eventual sequestration as amyloid can thus be seen as a protective response to block the toxic interface. We illustrate how the preferential self-association that gives this protection has been selectively favoured.

The influence of Cu2+ on the unfolding and refolding of intact and proteolytically processed β2‐microglobulin

Human beta(2)-microglobulin (beta(2)m) is an amyloidogenic protein in patients suffering from chronic kidney disease and especially in those patients that need intermittent hemodialysis for longer periods, e.g., when awaiting transplantation. While many in vitro conditions induce beta(2)m-amyloid formation from wild-type (wt) beta(2)m and while a number of structurally altered beta(2)m molecules are known to be conformationally unstable and amyloidogenic on their own, it is not known why beta(2)m-amyloid is generated in some dialysis patients. For many amyloid proteins it is known that divalent metal ions, especially Cu(2+), display strong binding and distinct destabilizing effects on protein conformation. The present study uses CE to assess conformational states of wt and cleaved beta(2)m (dK58-beta(2)m, beta(2)m cleaved at lysine-58, a modification found in the circulation of hemodialysis patients) in the presence of divalent metal ions. The experiments provide both qualitative and quantitative data showing the specific destabilizing effects of Cu(2+)-ions on the folding of wt beta(2)m. Both refolding after acid denaturation and solution structure of beta(2)m under otherwise native conditions are severely influenced by Cu(2+). An increased unfolding, aggregation, and induction of Congo red-reactive molecular species in Cu(2+)-incubated wt-beta(2)m could be demonstrated while the refolding kinetics of dK58-beta(2)m, already slower than the wt molecule, appeared not to be further decreased by Cu(2+). Given the interest in the actions of metal ions in other types of amyloidosis, including, e.g., Alzheimer's disease and the prion encephalopathies, the use of microelectrophoretic methods to monitor unfolding and refolding of biomolecules available in scarce amounts as shown in this study is an attractive option.

Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens

Many diverse human diseases are associated with protein aggregation in ordered fibrillar structures called amyloid. Amyloid formation may mediate aberrant protein interactions that culminate in neurodegeneration in Alzheimer, Huntington, and Parkinson diseases and in prion encephalopathies. Studies of protein aggregation in the brain are hampered by limitations in imaging techniques and often require invasive methods that can only be performed postmortem. Here we describe transgenic mice in which aggregation-prone proteins that cause Huntington and Parkinson disease are expressed in the ocular lens. Expression of a mutant huntingtin fragment or alpha-synuclein in the lens leads to protein aggregation and cataract formation, which can be monitored in real time by noninvasive, highly sensitive optical techniques. Expression of a mutant huntingtin fragment in mice lacking the major lens chaperone, alphaB-crystallin, markedly accelerated the onset and severity of aggregation, demonstrating that the endogenous chaperone activity of alphaB-crystallin suppresses aggregation in vivo. These novel mouse models will facilitate the characterization of protein aggregation in vivo and are being used in efficient and economical screens for chemical and genetic modifiers of disease-relevant protein aggregation.

Dimers Initiate and Propagate Serine Protease Inhibitor Polymerisation

The serine protease inhibitor (serpin) family can readily form long-chain polymers by a process that underlies a variety of diseases. We show here that monomers of plasma serpins α 1-antitrypsin and antithrombin are stable on incubation with the rate-limiting step in their polymerisation being the formation of the initial dimer. Once formed, the dimers readily interlink to form tetramers and can bind monomers to form trimers and longer oligomers. Cleavage of the only exposed reactive loop, in unit I of the dimers, prevents their interlinkage, but these cleaved dimers can still link to monomers. The rapid binding by the cleaved dimers of a peptide specific to the lower half of β-sheet A of the molecule indicates the ready opening of this β-sheet in unit II of the dimers. The failure of the cleaved dimers to bind peptide-complexed monomers, together with the relative inaccessibility of the P14 hinge residue in the oligomers, is evidence that partial insertion of the reactive loop into its own A-sheet is required for polymer formation. We propose that serpin dimers initiate and propagate polymerisation by having one exposed loop with an optimal conformation as a β-strand donor and a readily opened β-sheet as an acceptor. The sequential reformation of these activated β-interfaces as the oligomer extends, molecule by molecule, provides a model for the fibril and amyloid formation of conformational diseases in general as well as for the infectivity of prion encephalopathies.

Amino‐Terminally Truncated Prion Protein PrP90‐231 Induces Microglial Activation in Vitro

The conversion of the prion protein (PrP) into a protease-resistant isoform (PrP(Res)) is considered the pathogenic event responsible for prion encephalopathies. Microglia activation accompanies PrP(Res) deposition representing an early event in the progression of these diseases. It is now believed that microglial cells play a worsening, if not causative, role in prion-induced neuronal death, through the release of proinflammatory and neurotoxic molecules. Indeed, in vitro observations have demonstrated that PrP(Res) and the synthetic prion fragment PrP106-126 induce neuronal death by activating microglial to migrate in the lesion area and secrete cytokines. Recently, we and others have demonstrated that the recombinant peptide, corresponding to the protease-resistant portion of PrP encompassing the amino acids 90-231 (PrP90-231), when beta-structured, is toxic for neuronal cells, in vitro. Here we report that PrP90-231 induces activation of N9 microglial cells, characterized by cell proliferation arrest and increased secretion of different cytokines (RANTES, GCSF, and IL-12). Moreover, the treatment of N9 cells with PrP90-231 elicited inducible nitric oxide synthase (i-NOS) expression, nitric oxide release, and a delayed (15 min to 1 h of treatment) extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation/activation. Although ERK1/2 is known to regulate proliferative and differentiative events, we show that its blockade, using the specific MEK inhibitor PD98059, did not prevent PrP90-231-induced inhibition of N9 cell proliferation. To our knowledge, this is the first evidence that a recombinant PrP(Res)-like peptide elicits microglial activation in vitro, thus representing a potentially important tool to develop possible therapeutic strategies to target prion-induced brain inflammation.

Synergistic and strain-specific effects of bovine spongiform encephalopathy and scrapie prions in the cell-free conversion of recombinant prion protein

This study describes the conversion of murine PrP(C) by PrP(Sc) from three different mouse scrapie strains (ME7, 87V and 22A) and from a mouse-passaged bovine spongiform encephalopathy (BSE) strain (BSE/Bl6). This was demonstrated by a modified, non-radioactive, cell-free conversion assay using bacterial prion protein, which was converted into a proteinase K (PK)-resistant fragment designated PrP(res). Using this assay, newly formed PrP(res) could be detected by an antibody that discriminated de novo PrP(res) and the original PrP(Sc) seed. The results suggested that PrP(res) formation occurs in three phases: the first 48 h when PrP(res) formation is delayed, followed by a period of substantially accelerated PrP(res) formation and a plateau phase when a maximum concentration of PrP(res) is reached after 72 h. The conversion of prokaryotically expressed PrP(C) by ME7 and BSE prions led to unglycosylated, PK-digested, abnormal PrP(res) fragments, which differed in molecular mass by 1 kDa. Therefore, prion strain phenotypes were retained in the cell-free conversion, even when recombinant PrP(C) was used as the substrate. Moreover, co-incubation of ME7 and BSE prions resulted in equal amounts of both ME7- and BSE-derived PrP(res) fragments (as distinguished by their different molecular sizes) and also in a significantly increased total amount of de novo-generated PrP(res). This was found to be more than twice the amount of either strain when incubated separately. This result indicates a synergistic effect of both strains during cell-free conversion. It is not yet known whether such a cooperative action between BSE and scrapie prions also occurs in vivo.

Prions and retroviruses: an endosomal rendezvous?

The past two decades have seen the emergence of endocytic pathways as highly regulated systems for the sorting, selective degradation and recycling of nearly all cell‐surface membrane proteins. At the centre of these pathways are complex endosomal structures known as multivesicular bodies (MVBs) that house intraluminal vesicles (ILVs) formed by invagination and budding from the limiting membrane (Fig 1A). The ILVs contain membrane proteins from the Golgi complex and cell surface that are destined typically for degradation after the fusion of MVBs with lysosomes. This constitutive fate of ILVs is universal to eukaryotes and represents one of the main pathways for membrane protein degradation (Katzmann et al , 2002). Figure 1. Protein transport in the endo‐lysosomal pathways. ( A ) Topological relationships of the multivesicular body (MVB). Different types of cell‐surface proteins (indicated by blue and red molecules) can be internalized and transported by various routes (arrows) to endosomal compartments. The endosomal compartment is known as an MVB when its lumen contains intraluminal vesicles (ILVs) that are formed by budding from the limiting membrane. If the MVB fuses with the plasma membrane, ILVs are released into the extracellular space and become known as exosomes. ( B ) Transport into and out of the multivesicular body (MVB). Pathways linking the MVB with other compartments, such as the early endosome (EE), Golgi, lysosome and plasma membrane, are all regulated in a substrate‐specific and cell‐type‐specific manner to control the localization and fate of various membrane proteins (blue, red and green molecules). Alterations of these transport pathways is emerging as a key facet of viral pathogenesis and might also influence the intracellular accumulation and cell‐to‐cell transmission of prions. For example, adjacent cells, through interactions among cell surface signalling or adhesion molecules, might stimulate localized MVB fusion with the plasma membrane to facilitate exosome release and intercellular transfer of their cargo. …

Altered Glycosylation of Acetylcholinesterase in the Creutzfeldt-Jakob Cerebrospinal Fluid

Several neurodegenerative disorders present deficiencies in the cholinergic system. Scarce research on prion encephalopathies has examined the levels of cholinergic pathway-related enzymes. Acetylcholinesterase (AChE) is expressed as several molecular forms. The potential importance of these variants is increased by the possibility that AChE has roles other than acetylcholine hydrolysis. We investigated the levels of AChE, its molecular forms, and glycosylation in the cerebrospinal fluid (CSF) from Creutzfeldt-Jakob disease (CJD) patients.

Unsaturated Fatty Acids Induce Cytotoxic Aggregate Formation of Amyotrophic Lateral Sclerosis-linked Superoxide Dismutase 1 Mutants

Formation of misfolded protein aggregates is a remarkable hallmark of various neurodegenerative diseases including Alzheimer disease, Parkinson disease, Huntington disease, prion encephalopathies, and amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) immunoreactive inclusions have been found in the spinal cord of ALS animal models and patients, implicating the close involvement of SOD1 aggregates in ALS pathogenesis. Here we examined the molecular mechanism of aggregate formation of ALS-related SOD1 mutants in vitro. We found that long-chain unsaturated fatty acids (FAs) promoted aggregate formation of SOD1 mutants in both dose- and time-dependent manners. Metal-deficient SOD1s, wild-type, and mutants were highly oligomerized compared with holo-SOD1s by incubation in the presence of unsaturated FAs. Oligomerization of SOD1 is closely associated with its structural instability. Heat-treated holo-SOD1 mutants were readily oligomerized by the addition of unsaturated FAs, whereas wild-type SOD1 was not. The monounsaturated FA, oleic acid, directly bound to SOD1 and was characterized by a solid-phase FA binding assay using oleate-Sepharose. The FA binding characteristics were closely correlated with the oligomerization propensity of SOD1 proteins, which indicates that FA binding may change SOD1 conformation in a way that favors the formation of aggregates. High molecular mass aggregates of SOD1 induced by FAs have a granular morphology and show significant cytotoxicity. These findings suggest that SOD1 mutants gain FA binding abilities based on their structural instability and form cytotoxic granular aggregates.

Glycan-controlled epitopes of prion protein include a major determinant of susceptibility to sheep scrapie.

A key feature of prion encephalopathies is the accumulation of a misfolded form of the host glycoprotein PrP. Cell-free and cell culture studies have shown that the efficiency of conversion of PrP into the disease-associated form is influenced by its amino acid sequence and also by its carbohydrate moiety. Here, we characterize four novel glycoform-dependent monoclonal antibodies raised against prokaryotic recombinant sheep PrP. We demonstrate that these antibodies discriminate the PrP monoglycosylated species, since two of them recognize molecules that have the first Asn glycosylation site occupied (mono1) while the other two recognize molecules glycosylated at the second site (mono2). Remarkably, the recognition of PrP by the anti-mono2 antibodies was strongly influenced by the amino acid present at position 171, i.e., either Gln or Arg. This polymorphism is known to be the main determinant of susceptibility and resistance to scrapie in sheep. Altogether, our findings lead us to propose that each glycan chain controls the accessibility of PrP determinants located close upstream from their attachment site. The monoglycoform-assigned and the allotype-restricted antibodies described here, the first to date, should provide further opportunities to investigate the involvement of each glycan chain in PrP conversion in relation to prion strain diversity and the basis of the resistance conferred by the Arg-171 amino acid.

How Small Peptides Block and Reverse Serpin Polymerisation

Many of the late-onset dementias, including Alzheimer's disease and the prion encephalopathies, arise from the aberrant aggregation of individual proteins. The serpin family of serine protease inhibitors provides a well-defined structural example of such pathological aggregation, as its mutant variants readily form long-chain polymers, resulting in diseases ranging from thrombosis to dementia. The intermolecular linkages result from the insertion of the reactive site loop of one serpin molecule into the middle strand (s4A) position of the A beta-sheet of another molecule. We define here the structural requirements for small peptides to competitively bind to and block the s4A position to prevent this intermolecular linkage and polymerisation. The entry and anchoring of blocking-peptides is facilitated by the presence of a threonine which inserts into the site equivalent to P8 of s4A. But the critical requirement for small blocking-peptides is demonstrated in crystallographic structures of the complexes formed with selected tri- and tetrapeptides. These structures indicate that the binding is primarily due to the insertion of peptide hydrophobic side-chains into the P4 and P6 sites of s4A. The findings allow the rational design of synthetic blocking-peptides small enough to be suitable for mimetic design. This is demonstrated here with a tetrapeptide that preferentially blocks the polymerisation of a pathologically unstable serpin commonly present in people of European descent.

Cronopatología de las encefalopatías priónicas

Prion encephalopathies are a group of diseases with a hereditary or acquired origin which, after a long asymptomatic period, give rise to rapidly progressing neurological disorders. This progression can only be explained by an exponential growth of the pathogenic protein load, which allows to keep the load in low levels for many years and then to grow swiftly in a few months. Bearing in mind the knowledge currently available about the pathogenesis of prion diseases and patients' clinical progression, it becomes possible to distinguish several different periods of progression, the length of which can be estimated for each disease by reviewing the series of cases published to date. In general, the infectious prion diseases have a shorter period of latency than the hereditary ones and those caused by insertion of genetic material are associated to shorter latencies and to longer periods of illness than those caused by sporadic mutations. The rate of growth of the prion load depends essentially on how fast the pathogenic prion protein replicates; nevertheless, this growth is also modulated by other factors, many of which are polymorphisms in certain positions on the gene coding for prion protein or in other genes.

Preface [Hot Topic: Transmissible Spongiform Encephalopathies (Prion Diseases) Guest Editors: Xuemin Ye and Richard I. Carp

Preface [Hot Topic: Transmissible Spongiform Encephalopathies (Prion Diseases) Guest Editors: Xuemin Ye and Richard I. Carp

Preface [Hot Topic: Transmissible Spongiform Encephalopathies (Prion Diseases) Guest Editors: Xuemin Ye and Richard I. Carp

Preface [Hot Topic: Transmissible Spongiform Encephalopathies (Prion Diseases) Guest Editors: Xuemin Ye and Richard I. Carp

From Alzheimer to Huntington: why is a structural understanding so difficult?

An increasing family of neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases, prion encephalopathies and cystic fibrosis is associated with aggregation of misfolded polypeptide chains which are toxic to the cell. Knowledge of the three-dimensional structure of the proteins implicated is essential for understanding why and how endogenous proteins may adopt a non-native fold. Yet, structural work has been hampered by the difficulty of handling proteins insoluble or prone to aggregation, and at the same time that is why it is interesting to study these molecules. In this review, we compare the structural knowledge accumulated for two paradigmatic misfolding disorders, Alzheimer's disease (AD) and the family of poly-glutamine diseases (poly-Q) and discuss some of the hypotheses suggested for explaining aggregate formation. While a common mechanism between these pathologies remains to be proven, a direct comparison may help in designing new strategies for approaching their study.

Connecting the dots: trafficking of neurotrophins, lectins and diverse pathogens by binding to the neurotrophin receptor p75NTR.

The common receptor for neurotrophins, p75, has important roles in internalization and trafficking of neurotrophins along axons. Recent studies show that an astonishing array of proteins, including lectins, pathogens and neurotoxins, bind the p75 receptor, suggesting that they can hijack and utilize this receptor for trafficking between neuronal populations within the nervous system. Such pathogens include the neurologically important rabies viruses, prion proteins, beta-amyloid and possibly tetanus toxin. These proteins may hijack existing transport machineries designed to traffick neurotrophins, thus allowing the infiltration and distribution of pathogens and toxins among vulnerable neuronal populations with devastating effects, as seen in rabies, prion encephalopathies, Alzheimer's disease and tetanic muscle spasm. The discovery of an entry and transport machinery that is potentially shared between pathogens and neurotrophins sheds light ono trafficking systems in the nervous system and may assist the design of novel therapeutic avenues that prevent or slow the progression of diverse chronic and acute neurological disorders.

Serpinopathies and the conformational dementias.

The serpin superfamily of serine proteinase inhibitors has a central role in controlling proteinases in many biological pathways in a wide range of species. The inhibitory function of the serpins involves a marked conformational transition, but this inherent molecular flexibility also renders the serpins susceptible to point mutations that result in aberrant intermolecular linkage and polymer formation. The effects of such protein aggregation are cumulative, with a progressive loss of cellular function that results in diseases as diverse as cirrhosis and emphysema. The recent recognition that mutations in a serpin can also result in late-onset dementia provides insights into changes that underlie other conformational diseases, such as the amyloidoses, the prion encephalopathies and Huntington and Alzheimer diseases.