Cerebrospinal fluid lipoproteins inhibit α-synuclein aggregation by interacting with oligomeric species in seed amplification assays.

BACKGROUND: Aggregation of α-synuclein (α-syn) is a prominent feature of Parkinson's disease (PD) and other synucleinopathies. Currently, α-syn seed amplification assays (SAAs) using cerebrospinal fluid (CSF) represent the most promising diagnostic tools for synucleinopathies. However, CSF itself contains several compounds that can modulate the aggregation of α-syn in a patient-dependent manner, potentially undermining unoptimized α-syn SAAs and preventing seed quantification. METHODS: In this study, we characterized the inhibitory effect of CSF milieu on detection of α-syn aggregates by means of CSF fractionation, mass spectrometry, immunoassays, transmission electron microscopy, solution nuclear magnetic resonance spectroscopy, a highly accurate and standardized diagnostic SAA, and different in vitro aggregation conditions to evaluate spontaneous aggregation of α-syn. RESULTS: We found the high-molecular weight fraction of CSF (> 100,000 Da) to be highly inhibitory on α-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric α-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-α-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary α-syn intermediates. We observed significantly slower amplification of α-syn seeds in PD CSF when lipoproteins were added to the reaction mix of diagnostic SAA. Additionally, we observed a decreased inhibition capacity of CSF on α-syn aggregation after immunodepleting ApoA1 and ApoE. Finally, we observed that CSF ApoA1 and ApoE levels significantly correlated with SAA kinetic parameters in n = 31 SAA-negative control CSF samples spiked with preformed α-syn aggregates. CONCLUSIONS: Our results describe a novel interaction between lipoproteins and α-syn aggregates that inhibits the formation of α-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on α-syn aggregation explains the lack of quantitative results from analysis of SAA-derived kinetic parameters to date. Furthermore, our data show that lipoproteins are the main inhibitory components of CSF, suggesting that lipoprotein concentration measurements could be incorporated into data analysis models to eliminate the confounding effects of CSF milieu on α-syn quantification efforts.

Corrigendum: PMCA-based detection of prions in the olfactory mucosa of patients with sporadic Creutzfeldt-Jakob disease.

[This corrects the article DOI: 10.3389/fnagi.2022.848991.].

Approaching the Gut and Nasal Microbiota in Parkinson's Disease in the Era of the Seed Amplification Assays.

Parkinson's disease (PD) is a neurodegenerative disorder often associated with pre-motor symptoms involving both gastrointestinal and olfactory tissues. PD patients frequently suffer from hyposmia, hyposalivation, dysphagia and gastrointestinal dysfunctions. During the last few years it has been speculated that microbial agents could play a crucial role in PD. In particular, alterations of the microbiota composition (dysbiosis) might contribute to the formation of misfolded α-synuclein, which is believed to be the leading cause of PD. However, while several findings confirmed that there might be an important link between intestinal microbiota alterations and PD onset, little is known about the potential contribution of the nasal microbiota. Here, we describe the latest findings on this topic by considering that more than 80% of patients with PD develop remarkable olfactory deficits in their prodromal disease stage. Therefore, the nasal microbiota might contribute to PD, eventually boosting the gut microbiota in promoting disease onset. Finally, we present the applications of the seed amplification assays to the study of the gut and olfactory mucosa of PD patients, and how they could be exploited to investigate whether pathogenic bacteria present in the gut and the nose might promote α-synuclein misfolding and aggregation.

α-Synuclein Seed Amplification Assays for Diagnosing Synucleinopathies: The Way Forward.

Parkinson disease (PD) is the second most common neurodegenerative disease, and the most common synucleinopathy, as alpha-synuclein (α-syn), a prion-like protein, plays an important pathophysiologic role in its onset and progression. Although neuropathologic changes begin many years before the onset of motor manifestations, diagnosis still relies on the identification of the motor symptoms, which hinders to formulate an early diagnosis. Because α-syn misfolding and aggregation precede clinical manifestations, the possibility to identify these phenomena in patients with PD would allow us to recognize the disease at the earliest, premotor phases, as a consequence of the transition from a clinical to a molecular diagnosis. Seed amplification assays (SAAs) are a group of techniques that currently support the diagnosis of prion subacute encephalopathies, namely Creutzfeldt-Jakob disease. These techniques enable the detection of minimal amounts of prions in CSF and other matrices of affected patients. Recently, SAAs have been successfully applied to detect misfolded alpha-synuclein (α-syn) in CSF, olfactory mucosa, submandibular gland biopsies, skin, and saliva of patients with Parkinson disease (PD) and other synucleinopathies. In these categories, they can differentiate PD and dementia with Lewy bodies (DLBs) from control subjects, even in the prodromal stages of the disease. In differential diagnosis, SAAs satisfactorily differentiated PD, DLB, and multiple system atrophy (MSA) from nonsynucleinopathy parkinsonisms. The kinetic analysis of the SAA fluorescence profiles allowed the identification of synucleinopathy-dependent α-syn fibrils conformations, commonly referred to as strains, which have demonstrated diagnostic potential in differentiating among synucleinopathies, especially between Lewy body diseases (LBDs) (PD and DLB) and MSA. In front of these highly promising data, which make the α-syn seeding activity detected by SAAs as the most promising diagnostic biomarker for synucleinopathies, there are still preanalytical and analytical issues, mostly related to the assay standardization, which need to be solved. In this review, we discuss the key findings supporting the clinical application of α-syn SAAs to identify PD and other synucleinopathies, the unmet needs, and future perspectives.

PMCA-Based Detection of Prions in the Olfactory Mucosa of Patients With Sporadic Creutzfeldt-Jakob Disease.

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder caused by the conformational conversion of the prion protein (PrPC) into an abnormally folded form, named prion (or PrPSc). The combination of the polymorphism at codon 129 of the PrP gene (coding either methionine or valine) with the biochemical feature of the proteinase-K resistant PrP (generating either PrPSc type 1 or 2) gives rise to different PrPSc strains, which cause variable phenotypes of sCJD. The definitive diagnosis of sCJD and its classification can be achieved only post-mortem after PrPSc identification and characterization in the brain. By exploiting the Real-Time Quaking-Induced Conversion (RT-QuIC) assay, traces of PrPSc were found in the olfactory mucosa (OM) of sCJD patients, thus demonstrating that PrPSc is not confined to the brain. Here, we have optimized another technique, named protein misfolding cyclic amplification (PMCA) for detecting PrPSc in OM samples of sCJD patients. OM samples were collected from 27 sCJD and 2 genetic CJD patients (E200K). Samples from 34 patients with other neurodegenerative disorders were included as controls. Brains were collected from 26 sCJD patients and 16 of them underwent OM collection. Brain and OM samples were subjected to PMCA using the brains of transgenic mice expressing human PrPC with methionine at codon 129 as reaction substrates. The amplified products were analyzed by Western blot after proteinase K digestion. Quantitative PMCA was performed to estimate PrPSc concentration in OM. PMCA enabled the detection of prions in OM samples with 79.3% sensitivity and 100% specificity. Except for a few cases, a predominant type 1 PrPSc was generated, regardless of the tissues analyzed. Notably, all amplified PrPSc were less resistant to PK compared to the original strain. In conclusion, although the optimized PMCA did not consent to recognize sCJD subtypes from the analysis of OM collected from living patients, it enabled us to estimate for the first time the amount of prions accumulating in this biological tissue. Further assay optimizations are needed to faithfully amplify peripheral prions whose recognition could lead to a better diagnosis and selection of patients for future clinical trials.

Discrimination of MSA-P and MSA-C by RT-QuIC analysis of olfactory mucosa: the first assessment of assay reproducibility between two specialized laboratories.

BACKGROUND: Detection of the pathological and disease-associated alpha-synuclein (αSynD) in the brain is required to formulate the definitive diagnosis of multiple system atrophy (MSA) and Parkinson's disease (PD). We recently showed that αSynD can be detected in the olfactory mucosa (OM) of MSA and PD patients. For this reason, we have performed the first interlaboratory study based on α-synuclein Real-Time Quaking-Induced Conversion (αSyn_RT-QuIC) analysis of OM samples collected from PD and MSA patients with the parkinsonian (MSA-P) and cerebellar (MSA-C) phenotypes. METHODS: OM samples were prospectively collected from patients with a probable diagnosis of MSA-P (n = 20, mean disease duration 4.4 years), MSA-C (n = 10, mean disease duration 4 years), PD (n = 13, mean disease duration 8 years), and healthy control subjects (HS) (n = 11). Each sample was analyzed by αSyn_RT-QuIC in two independent specialized laboratories, one located in Italy (ITA-lab) and one located in the USA (USA-lab). Both laboratories have developed and used harmonized αSyn_RT-QuIC analytical procedures. Results were correlated with demographic and clinical data. RESULTS: The αSyn_RT-QuIC analysis reached a 96% interrater agreement of results (IAR) between laboratories (Kappa = 0.93, 95% CI 0.83-1.00). In particular, αSyn_RT-QuIC seeding activity was found in the OM of 9/13 patients with PD (sensitivity 69%, IAR 100%) and 18/20 patients with MSA-P (sensitivity 90%, IAR 100%). Interestingly, samples collected from patients with MSA-C did not induce αSyn_RT-QuIC seeding activity, except for one subject in USA-lab. Therefore, we found that MSA-P and MSA-C induced opposite effects. Regardless of disease diagnosis, the αSyn_RT-QuIC seeding activity correlated with some clinical parameters, including the rigidity and postural instability. CONCLUSIONS: Our study provides evidence that OM-αSynD may serve as a novel biomarker for accurate clinical diagnoses of PD, MSA-P, and MSA-C. Moreover, αSyn_RT-QuIC represents a reliable assay that can distinguish patients with MSA-P from those with MSA-C, and may lead to significant advancements in patients stratification and selection for emerging pharmacological treatments and clinical trials.

Sporadic Creutzfeldt-Jakob disease: Real-Time Quaking Induced Conversion (RT-QuIC) assay represents a major diagnostic advance.

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare and fatal neurodegenerative disorder with an incidence of 1.5 to 2 cases per million population/year. The disease is caused by a proteinaceous infectious agent, named prion (or PrPSc), which arises from the conformational conversion of the cellular prion protein (PrPC). Once formed, PrPSc interacts with the normally folded PrPC coercing it to undergo similar structural rearrangement. The disease is highly heterogeneous from a clinical and neuropathological point of view. The origin of this variability lies in the aberrant structures acquired by PrPSc. At least six different sCJD phenotypes have been described and each of them is thought to be caused by a peculiar PrPSc strain. Definitive sCJD diagnosis requires brain analysis with the aim of identifying intracerebral accumulation of PrPSc which currently represents the only reliable biomarker of the disease. Clinical diagnosis of sCJD is very challenging and is based on the combination of several clinical, instrumental and laboratory tests representing surrogate disease biomarkers. Thanks to the advent of the ultrasensitive Real-Time Quaking-Induced Conversion (RT-QuIC) assay, PrPSc was found in several peripheral tissues of sCJD patients, sometimes even before the clinical onset of the disease. This discovery represents an important step forward for the clinical diagnosis of sCJD. In this manuscript, we present an overview of the current applications and future perspectives of RT-QuIC in the field of sCJD diagnosis.

Animal Models of Autosomal Recessive Parkinsonism.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. The neuropathological hallmark of the disease is the loss of dopamine neurons of the substantia nigra pars compacta. The clinical manifestations of PD are bradykinesia, rigidity, resting tremors and postural instability. PD patients often display non-motor symptoms such as depression, anxiety, weakness, sleep disturbances and cognitive disorders. Although, in 90% of cases, PD has a sporadic onset of unknown etiology, highly penetrant rare genetic mutations in many genes have been linked with typical familial PD. Understanding the mechanisms behind the DA neuron death in these Mendelian forms may help to illuminate the pathogenesis of DA neuron degeneration in the more common forms of PD. A key step in the identification of the molecular pathways underlying DA neuron death, and in the development of therapeutic strategies, is the creation and characterization of animal models that faithfully recapitulate the human disease. In this review, we outline the current status of PD modeling using mouse, rat and non-mammalian models, focusing on animal models for autosomal recessive PD.

ß2-microglobulin triggers NLRP3 inflammasome activation in tumor-associated macrophages to promote multiple myeloma progression.

As substantial constituents of the multiple myeloma (MM) microenvironment, pro-inflammatory macrophages have emerged as key promoters of disease progression, bone destruction, and immune impairment. We identify beta-2-microglobulin (ß2m) as a driver in initiating inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed ß2m promotes ß2m amyloid aggregation in MAMs, resulting in lysosomal rupture and ultimately production of active interleukin-1ß (IL-1ß) and IL-18. This process depends on activation of the NLRP3 inflammasome after ß2m accumulation, as macrophages from NLRP3-deficient mice lack efficient ß2m-induced IL-1ß production. Moreover, depletion or silencing of ß2m in MM cells abrogates inflammasome activation in a murine MM model. Finally, we demonstrate that disruption of NLRP3 or IL-18 diminishes tumor growth and osteolytic bone destruction normally promoted by ß2m-induced inflammasome signaling. Our results provide mechanistic evidence for ß2m's role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 represents a potential therapeutic approach in MM.

PMCA-generated prions from the olfactory mucosa of patients with Fatal Familial Insomnia cause prion disease in mice.

Background: Fatal Familial Insomnia (FFI) is a genetic prion disease caused by the D178N mutation in the prion protein gene (PRNP) in coupling phase with methionine at PRNP 129. In 2017, we have shown that the olfactory mucosa (OM) collected from FFI patients contained traces of PrPSc detectable by Protein Misfolding Cyclic Amplification (PMCA). Methods: In this work, we have challenged PMCA-generated products obtained from OM and brain homogenate of FFI patients in BvPrP-Tg407 transgenic mice expressing the bank vole prion protein to test their ability to induce prion pathology. Results: All inoculated mice developed mild spongiform changes, astroglial activation, and PrPSc deposition mainly affecting the thalamus. However, their neuropathological alterations were different from those found in the brain of BvPrP-Tg407 mice injected with raw FFI brain homogenate. Conclusions: Although with some experimental constraints, we show that PrPSc present in OM of FFI patients is potentially infectious. Funding: This work was supported in part by the Italian Ministry of Health (GR-2013-02355724 and Ricerca Corrente), MJFF, ALZ, Alzheimer's Research UK and the Weston Brain Institute (BAND2015), and Euronanomed III (SPEEDY) to FM; by the Spanish Ministerio de Economía y Competitividad (grant AGL2016-78054-R [AEI/FEDER, UE]) to JMT and JCE; AM-M was supported by a fellowship from the INIA (FPI-SGIT-2015-02).

The Alpha-Synuclein RT-QuIC Products Generated by the Olfactory Mucosa of Patients with Parkinson's Disease and Multiple System Atrophy Induce Inflammatory Responses in SH-SY5Y Cells.

Parkinson's disease (PD) and multiple system atrophy (MSA) are caused by two distinct strains of disease-associated α-synuclein (αSynD). Recently, we have shown that olfactory mucosa (OM) samples of patients with PD and MSA can seed the aggregation of recombinant α-synuclein by means of Real-Time Quaking-Induced Conversion (αSyn_RT-QuIC). Remarkably, the biochemical and morphological properties of the final α-synuclein aggregates significantly differed between PD and MSA seeded samples. Here, these aggregates were given to neuron-like differentiated SH-SY5Y cells and distinct inflammatory responses were observed. To deepen whether the morphological features of α-synuclein aggregates were responsible for this variable SH-SY5Y inflammatory response, we generated three biochemically and morphologically distinct α-synuclein aggregates starting from recombinant α-synuclein that were used to seed αSyn_RT-QuIC reaction; the final reaction products were used to stimulate SH-SY5Y cells. Our study showed that, in contrast to OM samples of PD and MSA patients, the artificial aggregates did not transfer their distinctive features to the αSyn_RT-QuIC products and the latter induced analogous inflammatory responses in cells. Thus, the natural composition of the αSynD strains but also other specific factors in OM tissue can substantially modulate the biochemical, morphological and inflammatory features of the αSyn_RT-QuIC products.

Cell-free amplification of prions: Where do we stand?

Neurodegenerative diseases (NDs) such as Alzheimer's disease (AD), Parkinson's disease (PD), atypical parkinsonisms, frontotemporal dementia (FTLD) and prion diseases are characterized by the accumulation of misfolded proteins in the central nervous system (CNS). Although the cause for the initiation of protein aggregation is not well understood, these aggregates are disease-specific. For instance, AD is characterized by the intraneuronal accumulation of tau and extracellular deposition of amyloid-ß (Aß), PD is marked by the intraneuronal accumulation of α-synuclein, many FTLD are associated with the accumulation of TDP-43 while prion diseases show aggregates of misfolded prion protein. Hence, misfolded proteins are considered disease-specific biomarkers and their identification and localization in the CNS, collected postmortem, is required for a definitive diagnosis. With the development of two innovative cell-free amplification techniques named Protein Misfolding Cyclic Amplification (PMCA) and Real-Time Quaking-Induced Conversion (RT-QuIC), traces of disease-specific biomarkers were found in CSF and other peripheral tissues (e.g., urine, blood, and olfactory mucosa) of patients with different NDs. These techniques exploit an important feature shared by many misfolded proteins, that is their ability to interact with their normally folded counterparts and force them to undergo similar structural rearrangements. Essentially, RT-QuIC and PMCA mimic in vitro the same pathological processes of protein misfolding which occur in vivo in a very rapid manner. For this reason, they have been employed for studying different aspects of protein misfolding but, overall, they seem to be very promising for the premortem diagnosis of NDs.

Biochemical and biophysical comparison of human and mouse beta-2 microglobulin reveals the molecular determinants of low amyloid propensity.

The molecular bases of amyloid aggregation propensity are still poorly understood, especially for proteins that display a stable folded native structure. A prototypic example is human beta-2 microglobulin (ß2m), which, when accumulated in patients, gives rise to dialysis-related amyloidosis. Interestingly, although the physiologic concentration of ß2m in mice is five times higher than that found in human patients, no amyloid deposits are observed in mice. Moreover, murine ß2m (mß2m) not only displays a lower amyloid propensity both in vivo and in vitro but also inhibits the aggregation of human ß2m in vitro. Here, we compared human and mß2m for their aggregation propensity, ability to form soluble oligomers, stability, three-dimensional structure and dynamics. Our results indicate that mß2m low-aggregation propensity is due to two concomitant aspects: the low-aggregation propensity of its primary sequence combined with the absence of high-energy amyloid-competent conformations under native conditions. The identification of the specific properties determining the low-aggregation propensity of mouse ß2m will help delineate the molecular risk factors which cause a folded protein to aggregate.

Efficient RT-QuIC seeding activity for α-synuclein in olfactory mucosa samples of patients with Parkinson's disease and multiple system atrophy.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder whose diagnosis is often challenging because symptoms may overlap with neurodegenerative parkinsonisms. PD is characterized by intraneuronal accumulation of abnormal α-synuclein in brainstem while neurodegenerative parkinsonisms might be associated with accumulation of either α-synuclein, as in the case of Multiple System Atrophy (MSA) or tau, as in the case of Corticobasal Degeneration (CBD) and Progressive Supranuclear Palsy (PSP), in other disease-specific brain regions. Definite diagnosis of all these diseases can be formulated only neuropathologically by detection and localization of α-synuclein or tau aggregates in the brain. Compelling evidence suggests that trace-amount of these proteins can appear in peripheral tissues, including receptor neurons of the olfactory mucosa (OM). METHODS: We have set and standardized the experimental conditions to extend the ultrasensitive Real Time Quaking Induced Conversion (RT-QuIC) assay for OM analysis. In particular, by using human recombinant α-synuclein as substrate of reaction, we have assessed the ability of OM collected from patients with clinical diagnoses of PD and MSA to induce α-synuclein aggregation, and compared their seeding ability to that of OM samples collected from patients with clinical diagnoses of CBD and PSP. RESULTS: Our results showed that a significant percentage of MSA and PD samples induced α-synuclein aggregation with high efficiency, but also few samples of patients with the clinical diagnosis of CBD and PSP caused the same effect. Notably, the final RT-QuIC aggregates obtained from MSA and PD samples owned peculiar biochemical and morphological features potentially enabling their discrimination. CONCLUSIONS: Our study provide the proof-of-concept that olfactory mucosa samples collected from patients with PD and MSA possess important seeding activities for α-synuclein. Additional studies are required for (i) estimating sensitivity and specificity of the technique and for (ii) evaluating its application for the diagnosis of PD and neurodegenerative parkinsonisms. RT-QuIC analyses of OM and cerebrospinal fluid (CSF) can be combined with the aim of increasing the overall diagnostic accuracy of these diseases, especially in the early stages.

Prion Efficiently Replicates in α-Synuclein Knockout Mice.

Prion diseases are a group of neurodegenerative disorders associated with the conformational conversion of the cellular prion protein (PrPC) into an abnormal misfolded form named PrPSc. Other than accumulating in the brain, PrPSc can bind PrPC and force it to change conformation to PrPSc. The exact mechanism which underlies the process of PrPC/PrPSc conversion still needs to be defined and many molecules or cofactors might be involved. Several studies have documented an important role of PrPC to act as receptor for abnormally folded forms of α-synuclein which are responsible of a group of diseases known as synucleinopathies. The presence of PrPC was required to promote efficient internalization and spreading of abnormal α-synuclein between cells. In this work, we have assessed whether α-synuclein exerts any role in PrPSc conversion and propagation either in vitro or in vivo. Indeed, understanding the mechanism of PrPC/PrPSc conversion and the identification of cofactors involved in this process is crucial for developing new therapeutic strategies. Our results showed that PrPSc was able to efficiently propagate in the brain of animals even in the absence of α-synuclein thus suggesting that this protein did not act as key modulator of prion propagation. Thus, α-synuclein might take part in this process but is not specifically required for sustaining prion conversion and propagation.

Investigating the Molecular Basis of the Aggregation Propensity of the Pathological D76N Mutant of Beta-2 Microglobulin: Role of the Denatured State.

Beta-2 microglobulin (ß2m) is a protein responsible for a pathologic condition, known as dialysis-related amyloidosis (DRA), caused by its aggregation and subsequent amyloid formation. A naturally occurring mutation of ß2m, D76N, presents a higher amyloidogenic propensity compared to the wild type counterpart. Since the three-dimensional structure of the protein is essentially unaffected by the mutation, the increased aggregation propensity of D76N has been generally ascribed to its lower thermodynamic stability and increased dynamics. In this study we compare the equilibrium unfolding and the aggregation propensity of wild type ß2m and D76N variant at different experimental conditions. Our data revealed a surprising effect of the D76N mutation in the residual structure of the denatured state, which appears less compact than that of the wild type protein. A careful investigation of the structural malleability of the denatured state of wild type ß2m and D76N pinpoint a clear role of the denatured state in triggering the amyloidogenic propensity of the protein. The experimental results are discussed in the light of the previous work on ß2m and its role in disease.

Synthetic Prion Selection and Adaptation.

Prion pathologies are characterized by the conformational conversion of the cellular prion protein (PrPC) into a pathological infectious isoform, known as PrPSc. The latter acquires different abnormal conformations, which are associated with specific pathological phenotypes. Recent evidence suggests that prions adapt their conformation to changes in the context of replication. This phenomenon is known as either prion selection or adaptation, where distinct conformations of PrPSc with higher propensity to propagate in the new environment prevail over the others. Here, we show that a synthetically generated prion isolate, previously subjected to protein misfolding cyclic amplification (PMCA) and then injected in animals, is able to change its biochemical and biophysical properties according to the context of replication. In particular, in second transmission passage in vivo, two different prion isolates were found: one characterized by a predominance of the monoglycosylated band (PrPSc-M) and the other characterized by a predominance of the diglycosylated one (PrPSc-D). Neuropathological, biochemical, and biophysical assays confirmed that these PrPSc possess distinctive characteristics. Finally, PMCA analysis of PrPSc-M and PrPSc-D generated PrPSc (PrPSc-PMCA) whose biophysical properties were different from those of both inocula, suggesting that PMCA selectively amplified a third PrPSc isolate. Taken together, these results indicate that the context of replication plays a pivotal role in either prion selection or adaptation. By exploiting the ability of PMCA to mimic the process of prion replication in vitro, it might be possible to assess how changes in the replication environment influence the phenomenon of prion selection and adaptation.

Effects of peptidyl-prolyl isomerase 1 depletion in animal models of prion diseases.

Pin1 is a peptidyl-prolyl isomerase that induces the cis-trans conversion of specific Ser/Thr-Pro peptide bonds in phosphorylated proteins, leading to conformational changes through which Pin1 regulates protein stability and activity. Since down-regulation of Pin1 has been described in several neurodegenerative disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD), we investigated its potential role in prion diseases. Animals generated on wild-type (Pin1+/+), hemizygous (Pin1+/-) or knock-out (Pin1-/-) background for Pin1 were experimentally infected with RML prions. The study indicates that, neither the total depletion nor reduced levels of Pin1 significantly altered the clinical and neuropathological features of the disease.

Detection of prion seeding activity in the olfactory mucosa of patients with Fatal Familial Insomnia.

Fatal Familial Insomnia (FFI) is a genetic prion disease caused by a point mutation in the prion protein gene (PRNP) characterized by prominent thalamic atrophy, diffuse astrogliosis and moderate deposition of PrPSc in the brain. Here, for the first time, we demonstrate that the olfactory mucosa (OM) of patients with FFI contains trace amount of PrPSc detectable by PMCA and RT-QuIC. Quantitative PMCA analysis estimated a PrPSc concentration of about 1 × 10-14 g/ml. In contrast, PrPSc was not detected in OM samples from healthy controls and patients affected by other neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease and frontotemporal dementia. These results indicate that the detection limit of these assays is in the order of a single PrPSc oligomer/molecule with a specificity of 100%.