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. Electronic supplementary material The online version of this article (10.1186/s40035-019-0164-x) contains supplementary material, which is available to authorized users.

Prion diseases are neurodegenerative disorders which are caused by an accumulation of the abnormal, misfolded prion protein known as scrapie prion protein (PrPSc). These disorders are unique as they occur as sporadic, genetic and acquired forms. Sporadic Creutzfeldt-Jakob Disease (CJD) is the most common human prion disease, accounting for approximately 85–90% of cases, whereas autosomal dominant genetic forms, due to mutations in the prion protein gene (PRNP), account for 10–15% of cases. Genetic forms show a striking variability in their clinical and neuropathological picture and can sometimes mimic other neurodegenerative diseases.We report a novel PRNP mutation (V189I) in four CJD patients from three unrelated pedigrees. In three patients, the clinical features were typical for CJD and the diagnosis was pathologically confirmed, while the fourth patient presented with a complex phenotype including rapidly progressive dementia, behavioral abnormalities, ataxia and extrapyramidal features, and the diagnosis was probable CJD by current criteria, on the basis of PrPSc detection in CSF by Real Time Quaking-Induced Conversion assay. In all the three patients with autopsy findings, the neuropathological analysis revealed diffuse synaptic type deposition of proteinase K-resistant prion protein (PrPres), and type 1 PrPres was identified in the brain by western blot analysis. So, the histopathological and biochemical profile associated with the V189I mutation was indistinguishable from the MM1/MV1 subtype of sporadic CJD.Our findings support a pathogenic role for the V189I PRNP variant, confirm the heterogeneity of the clinical phenotypes associated to PRNP mutations and highlight the importance of PrPSc detection assays as diagnostic tools to unveil prion diseases presenting with atypical phenotypes.Electronic supplementary materialThe online version of this article (10.1186/s40478-018-0656-4) contains supplementary material, which is available to authorized users.

The conversion of the prion protein (PrPC) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrPC interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrPC constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrPC coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation.

The pathological deposition of the transactive response DNA-binding protein of 43 kDa (TDP-43) occurs in the majority (∼97%) of amyotrophic lateral sclerosis and in around 45% of frontotemporal lobar degeneration cases. Amyotrophic lateral sclerosis and frontotemporal lobar degeneration clinically overlap, presenting a continuum of phenotypes. Both amyotrophic lateral sclerosis and frontotemporal lobar degeneration lack treatments able to interfere with the underlying pathological process and early detection of TDP-43 pathology would facilitate the development of disease modifying drugs. The Real Time Quaking Induced Conversion reaction (RT-QuIC) showed the ability to detect prions in several peripheral tissues of patients with different forms of prion and prion-like diseases. Despite TDP-43 displays prion-like properties, to date the RT-QuIC technology has not yet been adapted to this protein. The aim of this study was to adapt the RT-QuIC technique for the TDP-43 substrate and to exploit the intrinsic ability of this technology to amplify minutes amount of misfolded proteins for the detection of pathological TDP-43 species in the CSF of amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients. We first optimized the technique with synthetic TDP-43 preformed aggregates and with autopsy-verified brain homogenate samples and subsequently analyzed CSF samples from amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients and controls. TDP-43 RT-QuIC was able to detect as little as 15 picograms of TDP-43 aggregates, discriminating between a cohort of subjects affected by amyotrophic lateral sclerosis and frontotemporal lobar degeneration and age-matched controls with a total sensitivity of 94% and a specificity of 85%. Our data give a proof-of-concept that TDP-43 is a suitable substrate for the RT-QuIC. TDP-43 RT-QuIC could be an innovative and useful tool for diagnosis and drug development in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. CSF detection of TDP-43 pathological aggregates may be exploited as a disease biomarker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients.

Prion, or PrPSc, is the pathological isoform of the cellular prion protein (PrPC) and it is the etiological agent of transmissible spongiform encephalopathies (TSE) affecting humans and animal species. The most relevant function of PrPC is its ability to bind copper ions through its flexible N-terminal moiety. This review includes an overview of the structure and function of PrPC with a focus on its ability to bind copper ions. The state-of-the-art of the role of copper in both PrPC physiology and in prion pathogenesis is also discussed. Finally, we describe the structural consequences of copper binding to the PrPC structure.

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%.

The cellular form of the prion protein (PrP C ) is a highly conserved glycoprotein mostly expressed in the central and peripheral nervous systems by different cell types in mammals. A misfolded, pathogenic isoform, denoted as prion, is related to a class of neurodegenerative diseases known as transmissible spongiform encephalopathy. PrP C function has not been unequivocally clarified, and it is rather defined as a pleiotropic protein likely acting as a dynamic cell surface scaffolding protein for the assembly of different signaling modules. Among the variety of PrP C protein interactors, the neuronal cell adhesion molecule (NCAM) has been studied in vivo, but the structural basis of this functional interaction is still a matter of debate. Here we focused on the structural determinants responsible for human PrP C (HuPrP) and NCAM interaction using stimulated emission depletion (STED) nanoscopy, SPR, and NMR spectroscopy approaches. PrP C co-localizes with NCAM in mouse hippocampal neurons, and this interaction is mainly mediated by the intrinsically disordered PrP C N-terminal tail, which binds with high affinity to the NCAM fibronectin type-3 domain. NMR structural investigations revealed surface-interacting epitopes governing the interaction between HuPrP N terminus and the second module of the NCAM fibronectin type-3 domain. Our data provided molecular details about the interaction between HuPrP and the NCAM fibronectin domain, and revealed a new role of PrP C N terminus as a dynamic and functional element responsible for protein-protein interaction.A misfolded form of the host-encoded cellular prion protein (PrP C ) 3 is the causative agent for a class of human and animal neurodegenerative diseases denoted as transmissible spongiform encephalopathies. PrP C is a sialoglycoprotein, tethered to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Soluble, natively ␣-helix-folded monomers of PrP C may adopt an aggregated protease-resistant conformation known as PrP Sc (1). The mature human PrP C (HuPrP) is composed of 209 residues including a largely unstructured N-terminal part and a globular ␣-helix-rich C-terminal domain (2). Conversely, PrP Sc is ␤-sheet-enriched, partially protease-resistant, insoluble, and multimeric (3). The insoluble nature of PrP Sc and its propensity to aggregate have hampered the use of high-resolution techniques, and therefore different PrP Sc models currently exist (4). Despite the fact that PrP C is highly conserved among different species, its physiological function has not been fully clarified. Defining PrP C function remains one of the main challenges in prion biology, and it is also an absolute requirement for understanding prion diseases. It is now being accepted that PrP C is a pivotal molecule with diverse roles in brain development and in neural plasticity in the adult (5-8). Proposed PrP C functions range from neuronal growth and differentiation (9), synaptic plasticity (10, 11), cell signaling (12, * This work was supported by the EC th...

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