Yeast prions form infectious amyloid inclusion bodies in bacteria

Background: Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. Results: Here we show that both the prion domain of Sup35 (Sup35-NM) and the Ure2 protein (Ure2p) form inclusion bodies (IBs) displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. Conclusions: An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity

Yeast prions form infectious amyloid inclusion bodies in bacteria

Background Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. Results Here we show that both the prion domain of Sup35 (Sup35-NM) and the Ure2 protein (Ure2p) form inclusion bodies (IBs) displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. Conclusions An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity

Tau Protein as a Biological Fluid Biomarker in Neurodegenerative Dementias

Tau is a microtubule-associated protein, whose main function is the modulation of the stability of axonal microtubules. In physiological conditions tau is abundant in neurons while its expression in glial populations is low and restricted to astrocytes and oligodendrocytes. The aggregation of tau in neurofibrillary or gliofibrillary tangles is the main hallmark of tauopathies, a complex group of human neurodegenerative conditions where tau hyper-phosphorylation causes its increased insolubility and aggregation leading to tangle formation and microtubule destabilization. Tau can be detected in biological fluids in physiological and pathological conditions. In several neurodegenerative dementias, either associated or not to a primary tauopathy, tau levels are altered in a disease-specific pattern, which can be used as a biomarker for disease diagnosis and prognosis. The study of tau levels in biological fluids has been mainly performed in the cerebrospinal fluid (CSF), although the recent development of ultrasensitive techniques allows the robust quantification of tau in blood-based biofluids such as serum and plasma. The presence of elevated total-tau in the CSF is assumed to reflect the degree of axonal damage in the brain tissue. Consequently, highest total-tau CSF levels are found in sporadic Creutzfeldt-Jakob disease, which is characterized by massive neuronal damage and a rapid progressive course. Elevated total-tau is also detected in Alzheimer’s disease and dementia with Lewy bodies, while in other dementia conditions such as vascular dementia, frontotemporal dementia and corticobasal degeneration are unchanged, inconclusive or not determined. Additionally, total-tau rises temporarily due to cerebral infarction. In contrast, elevated phospho-tau levels seem to be restricted to Alzheimer’s disease pathology, most likely mirroring the presence of the hyper-phosphorylated form in the brain tissue, although phospho-tau levels are mainly unaffected in tauopathies. Additionally, isoforms and different structural and truncated tau forms have also been reported to be altered in neurodegenerative dementias. In this complex scenario the diagnostic accuracy of diverse tau forms as disease-specific biomarkers needs to be established. In this chapter, we summarize the current knowledge on the alterations of diverse tau forms in biological fluids of neurodegenerative dementias and its relevance in the differential diagnostic context. Additionally, we explore how tau alterations in the brain tissue may explain the etiology of its regulated levels in CSF and blood

Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors

Background: The amyloid-β peptide (Aβ42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Aβ42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders. Results: Here we describe a rapid and high-throughput screening method to identify molecules able to modulate amyloid aggregation. The approach exploits the inclusion bodies (IBs) formed by Aβ42 when expressed in bacteria. We have shown previously that these aggregates retain amyloid structural and functional properties. In the present work, we demonstrate that their in vitro refolding is selectively sensitive to the presence of aggregation-promoting metal ions, allowing the detection of inhibitors of metal-promoted amyloid aggregation with potential therapeutic interest. Conclusions: Because IBs can be produced at high levels and easily purified, the method overcomes one of the main limitations in screens to detect amyloid modulators: the use of expensive and usually highly insoluble synthetic peptides

Amyloid-like protein inclusions in tobacco transgenic plants

The formation of insoluble protein deposits in human tissues is linked to the onset of more than 40 different disorders, ranging from dementia to diabetes. In these diseases, the proteins usually self-assemble into ordered β-sheet enriched aggregates known as amyloid fibrils. Here we study the structure of the inclusions formed by maize transglutaminase (TGZ) in the chloroplasts of tobacco transplastomic plants and demonstrate that they have an amyloid-like nature. Together with the evidence of amyloid structures in bacteria and fungi our data argue that amyloid formation is likely a ubiquitous process occurring across the different kingdoms of life. The discovery of amyloid conformations inside inclusions of genetically modified plants might have implications regarding their use for human applications

Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors

Background: The amyloid-β peptide (Aβ42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Aβ42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders. Results: Here we describe a rapid and high-throughput screening method to identify molecules able to modulate amyloid aggregation. The approach exploits the inclusion bodies (IBs) formed by Aβ42 when expressed in bacteria. We have shown previously that these aggregates retain amyloid structural and functional properties. In the present work, we demonstrate that their in vitro refolding is selectively sensitive to the presence of aggregation-promoting metal ions, allowing the detection of inhibitors of metal-promoted amyloid aggregation with potential therapeutic interest. Conclusions: Because IBs can be produced at high levels and easily purified, the method overcomes one of the main limitations in screens to detect amyloid modulators: the use of expensive and usually highly insoluble synthetic peptides

Optimization of the Real-Time Quaking-Induced Conversion Assay for Prion Disease Diagnosis

The real-time quaking-induced conversion (RT-QuIC) assay is a highly reproducible and robust methodology exhibiting an excellent pre-mortem diagnostic accuracy for prion diseases. However, the protocols might be time-consuming and improvement of the detection technology is needed. In the present study, we investigated the influence of a pre-analytical cerebrospinal fluid (CSF) treatment with proteinase K (PK) on the kinetic of the RT-QuIC signal response. For this purpose, we added PK at different concentrations in RT-QuIC reactions seeded with Creutzfeldt-Jakob disease (sCJD) CSF. We observed that a mild pre-analytical PK treatment of CSF samples resulted in an increased seeding efficiency of the RT-QuIC reaction. Quantitative seeding parameters, such as a higher area under the curve (AUC) value or a shorter lag phase indicated a higher conversion efficiency after treatment. The diagnostic accuracy resulting from 2 mu g/ml PK treatment was analyzed in a retrospective study, where we obtained a sensitivity of 89%. Additionally, we analyzed the agreement with the previously established standard RT-QuIC protocol without PK treatment in a prospective study. Here, we found an overall agreement of 94% to 96%. A Cohen's kappa of 0.9036 (95% CI: 0.8114-0.9958) indicates an almost perfect agreement between both protocols. In conclusion, the outcome of our study can be used for a further optimization of the RT-QuIC assay in particular for a reduction of the testing time

Detection of Cerebrospinal Fluid Neurofilament Light Chain as a Marker for Alpha-Synucleinopathies

Alpha-synucleinopathies, such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), are a class of neurodegenerative diseases. A diagnosis may be challenging because clinical symptoms partially overlap, and there is currently no reliable diagnostic test available. Therefore, we aimed to identify a suitable marker protein in cerebrospinal fluid (CSF) to distinguish either between different types of alpha-synucleinopathies or between alpha-synucleinopathies and controls. In this study, the regulation of different marker protein candidates, such as alpha-synuclein (a-Syn), neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and total tau (tau) in different types of alpha-synucleinopathies, had been analyzed by using an ultrasensitive test system called single-molecule array (SIMOA). Interestingly, we observed that CSF-NfL was significantly elevated in patients with DLB and MSA compared to patients with PD or control donors. To differentiate between groups, receiver operating characteristic (ROC) curve analysis resulted in a very good diagnostic accuracy as indicated by the area under the curve (AUC) values of 0.87-0.92 for CSF-NfL. Furthermore, we observed that GFAP and tau were slightly increased either in DLB or MSA, while a-Syn levels remained unregulated. Our study suggests NfL as a promising marker to discriminate between different types of alpha-synucleinopathies or between DLB/MSA and controls

Effect of the micro-environment on alpha-synuclein conversion and implication in seeded conversion assays

Background: α-Synuclein is a small soluble protein, whose physiological function in the healthy brain is poorly understood. Intracellular inclusions of α-synuclein, referred to as Lewy bodies (LBs), are pathological hallmarks of α- synucleinopathies, such as Parkinson’s disease (PD) or dementia with Lewy bodies (DLB). Main body: Understanding of the molecular basis as well as the factors or conditions promoting α-synuclein misfolding and aggregation is an important step towards the comprehension of pathological mechanism of α- synucleinopathies and for the development of efficient therapeutic strategies. Based on the conversion and aggregation mechanism of α-synuclein, novel diagnostic tests, such as protein misfolding seeded conversion assays, e.g. the real-time quaking-induced conversion (RT-QuIC), had been developed. In diagnostics, α-synuclein RT-QuIC exhibits a specificity between 82 and 100% while the sensitivity varies between 70 and 100% among different laboratories. In addition, the α-synuclein RT-QuIC can be used to study the α-synuclein-seeding-characteristics of different α-synucleinopathies and to differentiate between DLB and PD. Conclusion: The variable diagnostic accuracy of current α-synuclein RT-QuIC occurs due to different protocols, cohorts and material etc.. An impact of micro-environmental factors on the α-synuclein aggregation and conversion process and the occurrence and detection of differential misfolded α-synuclein types or strains might underpin the clinical heterogeneity of α-synucleinopathies

Plasma YKL-40 in the spectrum of neurodegenerative dementia

Background: Increased plasma YKL-40 has been reported in Alzheimer's disease (AD), but its levels in other neurodegenerative diseases are unknown. Here, we aimed to investigate plasma YKL-40 in the spectrum of neurodegenerative dementias. Methods: YKL-40 was quantified in the plasma of 315 cases, including healthy controls (HC), neurological disease controls (ND), AD, vascular dementia (VaD), frontotemporal dementia (FTD), sporadic Creutzfeldt-Jakob disease (CJD) and Lewy body dementia (LBD). Diagnostic accuracy in the differential diagnostic context and influence of age and gender was assessed. Results: Highest YKL-40 levels were detected in CJD, followed by LBD, VaD, AD, FTD, ND and HC. YKL-40 was associated to age but not to sex. After controlling for age, YKL-40 was significantly elevated in CJD compared to HC (p<0.001), ND, AD and VaD (p<0.01) and in LBD compared to HC (p<0.05). In CJD, YKL-40 concentrations were significantly higher at late disease stages. Conclusions: Plasma YKL-40 is significantly elevated in CJD regardless of clinical and genetic parameters, with moderate diagnostic accuracy in the discrimination from control cases. Our study discards a potential use of this biomarker in the differential diagnostic context but opens the possibility to be explored as a marker for CJD monitoring