Detection of TDP-43 seeding activity in the olfactory mucosa from patients with frontotemporal dementia.

INTRODUCTION: We assessed TAR DNA-binding protein 43 (TDP-43) seeding activity and aggregates detection in olfactory mucosa of patients with frontotemporal lobar degeneration with TDP-43-immunoreactive pathology (FTLD-TDP) by TDP-43 seeding amplification assay (TDP43-SAA) and immunocytochemical analysis. METHODS: The TDP43-SAA was optimized using frontal cortex samples from 16 post mortem cases with FTLD-TDP, FTLD with tau inclusions, and controls. Subsequently, olfactory mucosa samples were collected from 17 patients with FTLD-TDP, 15 healthy controls, and three patients carrying MAPT variants. RESULTS: TDP43-SAA discriminated with 100% accuracy post mortem cases presenting or lacking TDP-43 neuropathology. TDP-43 seeding activity was detectable in the olfactory mucosa, and 82.4% of patients with FTLD-TDP tested positive, whereas 86.7% of controls tested negative (P < 0.001). Two out of three patients with MAPT mutations tested negative. In TDP43-SAA positive samples, cytoplasmatic deposits of phosphorylated TDP-43 in the olfactory neural cells were detected. DISCUSSION: TDP-43 aggregates can be detectable in olfactory mucosa, suggesting that TDP43-SAA might be useful for identifying and monitoring FTLD-TDP in living patients.

Different tau fibril types reduce prion level in chronically and de novo infected cells.

Neurodegenerative diseases are often characterized by the codeposition of different amyloidogenic proteins, normally defining distinct proteinopathies. An example is represented by prion diseases, where the classical deposition of the aberrant conformational isoform of the prion protein (PrPSc) can be associated with tau insoluble species, which are usually involved in another class of diseases called tauopathies. How this copresence of amyloidogenic proteins can influence the progression of prion diseases is still a matter of debate. Recently, the cellular form of the prion protein, PrPC, has been investigated as a possible receptor of amyloidogenic proteins, since its binding activity with Aß, tau, and α-synuclein has been reported, and it has been linked to several neurotoxic behaviors exerted by these proteins. We have previously shown that the treatment of chronically prion-infected cells with tau K18 fibrils reduced PrPSc levels. In this work, we further explored this mechanism by using another tau construct that includes the sequence that forms the core of Alzheimer's disease tau filaments in vivo to obtain a distinct fibril type. Despite a difference of six amino acids, these two constructs form fibrils characterized by distinct biochemical and biological features. However, their effects on PrPSc reduction were comparable and probably based on the binding to PrPC at the plasma membrane, inhibiting the pathological conversion event. Our results suggest PrPC as receptor for different types of tau fibrils and point out a role of tau amyloid fibrils in preventing the pathological PrPC to PrPSc conformational change.

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.].

Copper coordination modulates prion conversion and infectivity in mammalian prion proteins.

In mammals the cellular form of the prion protein (PrPC) is a ubiquitous protein involved in many relevant functions in the central nervous system. In addition to its physiological functions PrPC plays a central role in a group of invariably fatal neurodegenerative disorders collectively called prion diseases. In fact, the protein is a substrate in a process in which it converts into an infectious and pathological form denoted as prion. The protein has a unique primary structure where the unstructured N-terminal moiety possesses characteristic sequences wherein histidines are able to coordinate metal ions, in particular copper ions. These sequences are called octarepeats for their characteristic length. Moreover, a non-octarepeat fifth-copper binding site is present where copper coordination seems to control infectivity. In this review, I will argue that these sequences may play a significant role in modulating prion conversion and replication.

Therapeutic strategies for identifying small molecules against prion diseases.

Prion diseases are fatal neurodegenerative disorders, for which there are no effective therapeutic and diagnostic agents. The main pathological hallmark has been identified as conformational changes of the cellular isoform prion protein (PrPC) to a misfolded isoform of the prion protein (PrPSc). Targeting PrPC and its conversion to PrPSc is still the central dogma in prion drug discovery, particularly in in silico and in vitro screening endeavors, leading to the identification of many small molecules with therapeutic potential. Nonetheless, multiple pathological targets are critically involved in the intricate pathogenesis of prion diseases. In this context, multi-target-directed ligands (MTDLs) emerge as valuable therapeutic approach for their potential to effectively counteract the complex etiopathogenesis by simultaneously modulating multiple targets. In addition, diagnosis occurs late in the disease process, and consequently a successful therapeutic intervention cannot be provided. In this respect, small molecule theranostics, which combine imaging and therapeutic properties, showed tremendous potential to cure and diagnose in vivo prion diseases. Herein, we review the major advances in prion drug discovery, from anti-prion small molecules identified by means of in silico and in vitro screening approaches to two rational strategies, namely MTDLs and theranostics, that have led to the identification of novel compounds with an expanded anti-prion profile.

Bifunctional carbazole derivatives for simultaneous therapy and fluorescence imaging in prion disease murine cell models.

Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrPSc) of the cellular prion protein (PrPC). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrPC conformation and prevents its pathogenic conversion. A focused library was synthesized via a four-step route, and a representative member was confirmed to have native fluorescence, including a band in the near-infrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrPSc. Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrPC levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrPSc aggregates by 2b was confirmed in living cells. 2b emerges as an initial hit compound for further medicinal chemistry optimization towards strain-independent anti-prion compounds.

Neuronal haemoglobin induces loss of dopaminergic neurons in mouse Substantia nigra, cognitive deficits and cleavage of endogenous α-synuclein.

Parkinson's disease (PD) presents the selective loss of A9 dopaminergic (DA) neurons of Substantia Nigra pars compacta (SNpc) and the presence of intracellular aggregates called Lewy bodies. α-synuclein (α-syn) species truncated at the carboxy-terminal (C-terminal) accumulate in pathological inclusions and promote α-syn aggregation and toxicity. Haemoglobin (Hb) is the major oxygen carrier protein in erythrocytes. In addition, Hb is expressed in A9 DA neurons where it influences mitochondrial activity. Hb overexpression increases cells' vulnerability in a neurochemical model of PD in vitro and forms cytoplasmic and nucleolar aggregates upon short-term overexpression in mouse SNpc. In this study, α and ß-globin chains were co-expressed in DA cells of SNpc in vivo upon stereotaxic injections of an Adeno-Associated Virus isotype 9 (AAV9) and in DA iMN9D cells in vitro. Long-term Hb over-expression in SNpc induced the loss of about 50% of DA neurons, mild motor impairments, and deficits in recognition and spatial working memory. Hb triggered the formation of endogenous α-syn C-terminal truncated species. Similar α-syn fragments were found in vitro in DA iMN9D cells over-expressing α and ß- globins when treated with pre-formed α-syn fibrils. Our study positions Hb as a relevant player in PD pathogenesis for its ability to trigger DA cells' loss in vivo and the formation of C-terminal α-syn fragments.

Discovery of Dual Aß/Tau Inhibitors and Evaluation of Their Therapeutic Effect on a Drosophila Model of Alzheimer's Disease.

Alzheimer's disease (AD), the most common type of dementia, currently represents an extremely challenging and unmet medical need worldwide. Amyloid-ß (Aß) and Tau proteins are prototypical AD hallmarks, as well as validated drug targets. Accumulating evidence now suggests that they synergistically contribute to disease pathogenesis. This could not only help explain negative results from anti-Aß clinical trials but also indicate that therapies solely directed at one of them may have to be reconsidered. Based on this, herein, we describe the development of a focused library of 2,4-thiazolidinedione (TZD)-based bivalent derivatives as dual Aß and Tau aggregation inhibitors. The aggregating activity of the 24 synthesized derivatives was tested in intact Escherichia coli cells overexpressing Aß42 and Tau proteins. We then evaluated their neuronal toxicity and ability to cross the blood-brain barrier (BBB), together with the in vitro interaction with the two isolated proteins. Finally, the most promising (most active, nontoxic, and BBB-permeable) compounds 22 and 23 were tested in vivo, in a Drosophila melanogaster model of AD. The carbazole derivative 22 (20 µM) showed extremely encouraging results, being able to improve both the lifespan and the climbing abilities of Aß42 expressing flies and generating a better outcome than doxycycline (50 µM). Moreover, 22 proved to be able to decrease Aß42 aggregates in the brains of the flies. We conclude that bivalent small molecules based on 22 deserve further attention as hits for dual Aß/Tau aggregation inhibition in AD.

Gerstmann-Sträussler-Scheinker Disease with F198S Mutation Induces Independent Tau and Prion Protein Pathologies in Bank Voles.

Gerstmann-Sträussler-Scheinker disease (GSS) is a rare genetic prion disease. A large GSS kindred linked to the serine-for-phenylalanine substitution at codon 198 of the prion protein gene (GSS-F198S) is characterized by conspicuous accumulation of prion protein (PrP)-amyloid deposits and neurofibrillary tangles. Recently, we demonstrated the transmissibility of GSS-F198S prions to bank vole carrying isoleucine at 109 PrP codon (BvI). Here we investigated: (i) the transmissibility of GSS-F198S prions to voles carrying methionine at codon 109 (BvM); (ii) the induction of hyperphosphorylated Tau (pTau) in two vole lines, and (iii) compared the phenotype of GSS-F198S-induced pTau with pTau induced in BvM following intracerebral inoculation of a familial Alzheimer's disease case carrying Presenilin 1 mutation (fAD-PS1). We did not detect prion transmission to BvM, despite the high susceptibility of BvI previously observed. Immunohistochemistry established the presence of induced pTau depositions in vole brains that were not affected by prions. Furthermore, the phenotype of pTau deposits in vole brains was similar in GSS-F198S and fAD-PS1. Overall, results suggest that, regardless of the cause of pTau deposition and its relationship with PrPSc in GSS-F198S human-affected brains, the two components possess their own seeding properties, and that pTau deposition is similarly induced by GSS-F198S and fAD-PS1.

Structural and dynamical determinants of a ß-sheet-enriched intermediate involved in amyloid fibrillar assembly of human prion protein.

The conformational conversion of the cellular prion protein (PrPC) into a misfolded, aggregated and infectious scrapie isoform is associated with prion disease pathology and neurodegeneration. Despite the significant number of experimental and theoretical studies the molecular mechanism regulating this structural transition is still poorly understood. Here, via Nuclear Magnetic Resonance (NMR) methodologies we investigate at the atomic level the mechanism of the human HuPrP(90-231) thermal unfolding and characterize the conformational equilibrium between its native structure and a ß-enriched intermediate state, named ß-PrPI. By comparing the folding mechanisms of metal-free and Cu2+-bound HuPrP(23-231) and HuPrP(90-231) we show that the coupling between the N- and C-terminal domains, through transient electrostatic interactions, is the key molecular process in tuning long-range correlated µs-ms dynamics that in turn modulate the folding process. Moreover, via thioflavin T (ThT)-fluorescence fibrillization assays we show that ß-PrPI is involved in the initial stages of PrP fibrillation, overall providing a clear molecular description of the initial phases of prion misfolding. Finally, we show by using Real-Time Quaking-Induced Conversion (RT-QuIC) that the ß-PrPI acts as a seed for the formation of amyloid aggregates with a seeding activity comparable to that of human infectious prions.

Evidence of Orientation-Dependent Early States of Prion Protein Misfolded Structures from Single Molecule Force Spectroscopy.

Prion diseases are neurodegenerative disorders characterized by the presence of oligomers and amyloid fibrils. These are the result of protein aggregation processes of the cellular prion protein (PrPC) into amyloidal forms denoted as prions or PrPSc. We employed atomic force microscopy (AFM) for single molecule pulling (single molecule force spectroscopy, SMFS) experiments on the recombinant truncated murine prion protein (PrP) domain to characterize its conformations and potential initial oligomerization processes. Our AFM-SMFS results point to a complex scenario of structural heterogeneity of PrP at the monomeric and dimer level, like other amyloid proteins involved in similar pathologies. By applying this technique, we revealed that the PrP C-terminal domain unfolds in a two-state process. We used two dimeric constructs with different PrP reciprocal orientations: one construct with two sequential PrP in the N- to C-terminal orientation (N-C dimer) and a second one in the C- to C-terminal orientation (C-C dimer). The analysis revealed that the different behavior in terms of unfolding force, whereby the dimer placed C-C dimer unfolds at a higher force compared to the N-C orientation. We propose that the C-C dimer orientation may represent a building block of amyloid fibril formation.

Recent advances in cellular models for discovering prion disease therapeutics.

INTRODUCTION: Prion diseases are a group of rare and lethal, rapidly progressive neurodegenerative diseases arising due to conversion of the physiological cellular prion protein into its pathological counterparts, denoted as 'prions.' These agents are resistant to inactivation by standard decontamination procedures and can be transmitted between individuals, consequently driving the irreversible brain damage typical of the diseases. AREAS COVERED: Since its infancy, prion research has mainly depended on animal models for untangling the pathogenesis of the disease as well as for the drug development studies. With the advent of prion-infected cell lines, relevant animal models have been complemented by a variety of cell-based models presenting a much faster, ethically acceptable alternative. EXPERT OPINION: To date, there are still either no effective prophylactic regimens or therapies for human prion diseases. Therefore, there is an urgent need for more relevant cellular models that best approximate in vivo models. Each cellular model presented and discussed in detail in this review has its own benefits and limitations. Once embarking in a drug screening campaign for the identification of molecules that could interfere with prion conversion and replication, one should carefully consider the ideal cellular model.

Innovative Non-PrP-Targeted Drug Strategy Designed to Enhance Prion Clearance.

Prion diseases are a group of neurodegenerative disorders characterized by the accumulation of misfolded prion protein (called PrPSc). Although conversion of the cellular prion protein (PrPC) to PrPSc is still not completely understood, most of the therapies developed until now are based on blocking this process. Here, we propose a new drug strategy aimed at clearing prions without any direct interaction with neither PrPC nor PrPSc. Starting from the recent discovery of SERPINA3/SerpinA3n upregulation during prion diseases, we have identified a small molecule, named compound 5 (ARN1468), inhibiting the function of these serpins and effectively reducing prion load in chronically infected cells. Although the low bioavailability of this compound does not allow in vivo studies in prion-infected mice, our strategy emerges as a novel and effective approach to the treatment of prion disease.

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.

Serpin Signatures in Prion and Alzheimer's Diseases.

Serpins represent the most broadly distributed superfamily of proteases inhibitors. They contribute to a variety of physiological functions and any alteration of the serpin-protease equilibrium can lead to severe consequences. SERPINA3 dysregulation has been associated with Alzheimer's disease (AD) and prion diseases. In this study, we investigated the differential expression of serpin superfamily members in neurodegenerative diseases. SERPIN expression was analyzed in human frontal cortex samples from cases of sporadic Creutzfeldt-Jakob disease (sCJD), patients at early stages of AD-related pathology, and age-matched controls not affected by neurodegenerative disorders. In addition, we studied whether Serpin expression was dysregulated in two animal models of prion disease and AD.Our analysis revealed that, besides the already observed upregulation of SERPINA3 in patients with prion disease and AD, SERPINB1, SERPINB6, SERPING1, SERPINH1, and SERPINI1 were dysregulated in sCJD individuals compared to controls, while only SERPINB1 was upregulated in AD patients. Furthermore, we analyzed whether other serpin members were differentially expressed in prion-infected mice compared to controls and, together with SerpinA3n, SerpinF2 increased levels were observed. Interestingly, SerpinA3n transcript and protein were upregulated in a mouse model of AD. The SERPINA3/SerpinA3nincreased anti-protease activity found in post-mortem brain tissue of AD and prion disease samples suggest its involvement in the neurodegenerative processes. A SERPINA3/SerpinA3n role in neurodegenerative disease-related protein aggregation was further corroborated by in vitro SerpinA3n-dependent prion accumulation changes. Our results indicate SERPINA3/SerpinA3n is a potential therapeutic target for the treatment of prion and prion-like neurodegenerative diseases.

The E3 Ubiquitin Ligase TRAF6 Interacts with the Cellular Prion Protein and Modulates Its Solubility and Recruitment to Cytoplasmic p62/SQSTM1-Positive Aggresome-Like Structures.

The cellular prion protein (PrPC) is a ubiquitous glycoprotein highly expressed in the brain where it is involved in neurite outgrowth, copper homeostasis, NMDA receptor regulation, cell adhesion, and cell signaling. Conformational conversion of PrPC into its insoluble and aggregation-prone scrapie form (PrPSc) is the trigger for several rare devastating neurodegenerative disorders, collectively referred to as prion diseases. Recent work indicates that the ubiquitin-proteasome system is involved in quality control of PrPC. To better dissect the role of ubiquitination in PrPC physiology, we focused on the E3 RING ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here, we report that PrPC interacts with TRAF6 both in vitro, in cells, and in vivo, in the mouse brain. Transient overexpression of TRAF6 indirectly modulates PrPC ubiquitination and triggers redistribution of PrPC into the insoluble fraction. Importantly, in the presence of wild-type TRAF6, but not a mutant lacking E3 ligase activity, PrPC accumulates into cytoplasmic aggresome-like inclusions containing TRAF6 and p62/SQSTM1. Our results suggest that TRAF6 ligase activity could exert a role in the regulation of PrPC redistribution in cells under physiological conditions. This novel interaction may uncover possible mechanisms of cell clearance/reorganization in prion diseases.

Expression pattern of perilipins in human brain during aging and in Alzheimer's disease.

AIMS: Perilipins are conserved proteins that decorate intracellular lipid droplets and are essential for lipid metabolism. To date, there is limited knowledge on their expression in human brain or their involvement in brain aging and neurodegeneration. The aim of this study was to characterise the expression levels of perilipins (Plin1-Plin5) in different cerebral areas from subjects of different age, with or without signs of neurodegeneration. METHODS: We performed real-time RT-PCR, western blotting, immunohistochemistry and confocal microscopy analyses in autoptic brain samples of frontal and temporal cortex, cerebellum and hippocampus from subjects ranging from 33 to 104 years of age, with or without histological signs of neurodegeneration. To test the possible relationship between Plins and inflammation, correlation analysis with IL-6 expression was also performed. RESULTS: Plin2, Plin3 and Plin5, but not Plin1 and Plin4, are expressed in the considered brain areas with different intensities. Plin2 appears to be expressed more in grey matter, particularly in neurons in all the areas analysed, whereas Plin3 and Plin5 appear to be expressed more in white matter. Plin3 seems to be expressed more in astrocytes. Only Plin2 expression is higher in old subjects and patients with early tauopathy or Alzheimer's disease and is associated with IL-6 expression. CONCLUSIONS: Perilipins are expressed in human brain but only Plin2 appears to be modulated with age and neurodegeneration and linked to an inflammatory state. We propose that the accumulation of lipid droplets decorated with Plin2 occurs during brain aging and that this accumulation may be an early marker and initial step of inflammation and neurodegeneration.

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.

The Cellular Prion Protein Increases the Uptake and Toxicity of TDP-43 Fibrils.

Cytoplasmic aggregation of the primarily nuclear TAR DNA-binding protein 43 (TDP-43) affects neurons in most amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal lobar degeneration (FTLD) cases. The cellular prion protein, PrPC, has been recognized as a common receptor and downstream effector of circulating neurotoxic species of several proteins involved in neurodegeneration. Here, capitalizing on our recently adapted TDP-43 real time quaking induced reaction, we set reproducible protocols to obtain standardized preparations of recombinant TDP-43 fibrils. We then exploited two different cellular systems (human SH-SY5Y and mouse N2a neuroblastoma cells) engineered to express low or high PrPC levels to investigate the link between PrPC expression on the cell surface and the internalization of TDP-43 fibrils. Fibril uptake was increased in cells overexpressing either human or mouse prion protein. Increased internalization was associated with detrimental consequences in all PrP-overexpressing cell lines but was milder in cells expressing the human form of the prion protein. As described for other amyloids, treatment with TDP-43 fibrils induced a reduction in the accumulation of the misfolded form of PrPC, PrPSc, in cells chronically infected with prions. Our results expand the list of misfolded proteins whose uptake and detrimental effects are mediated by PrPC, which encompass almost all pathological amyloids involved in neurodegeneration.