Abstract
Experimental autoimmune encephalomyelitis (EAE) is the most commonly used experimental model for human multiple sclerosis, an autoimmune disease driven by differentiated Th1 and Th17 cells, ...and inflammatory dendritic cells (DCs). Recent data show that EAE is worsened in mice lacking PrPC protein and disease exacerbation has been attributed to T cells, which would differentiate into more aggressive effectors, when deprived of PrPC.
Novel compounds, modulating PrPC activity, have been recently developed, although not tested in EAE models yet. Interestingly, by specific gene array studies, we found that PrPC was highly expressed in DCs and differentially expressed among selected DC subsets. Moreover, by using a series of computational, and biochemical assays we identified novel small molecules, able to modulate PrPC function in specific cell lines overexpressing PrPC. By employing such tools, we studied the impact of PrPC modulation in promoting regulatory DC subsets. To this aim, DCs were treated either with novel PrPC modulators or a reference PrPC binding molecule Fe (III)-TMPyP. Then, they were co-cultured with sorted naïve CD4+ T cells in vitro. Notably, we found a significant expansion of FOXP3+CD4+ T regulatory (Treg) cells in cultures containing DC subsets, which were pre-treated with the specific PrPC modulators. Moreover, in a EAE model, we showed that systemic administration of such PrPC regulators, resulted in significant reduction of disease severity, compared to untreated controls. Overall, our results suggest that PrPC modulation in selected DC subsets may represent a novel means to restrain inflammatory T cell effectors, resulting in reduced inflammation, demyelination, and axonal injury in a model of EAE.
Aggregation and cytoplasmic mislocalization of TDP-43 are pathological hallmarks of amyotrophic lateral sclerosis and frontotemporal dementia spectrum. However, the molecular mechanism by which ...TDP-43 aggregates form and cause neurodegeneration remains poorly understood. Cyclophilin A, also known as peptidyl-prolyl cis-trans isomerase A (PPIA), is a foldase and molecular chaperone. We previously found that PPIA interacts with TDP-43 and governs some of its functions, and its deficiency accelerates disease in a mouse model of amyotrophic lateral sclerosis. Here we characterized PPIA knock-out mice throughout their lifespan and found that they develop a neurodegenerative disease with key behavioural features of frontotemporal dementia, marked TDP-43 pathology and late-onset motor dysfunction. In the mouse brain, deficient PPIA induces mislocalization and aggregation of the GTP-binding nuclear protein Ran, a PPIA interactor and a master regulator of nucleocytoplasmic transport, also for TDP-43. Moreover, in absence of PPIA, TDP-43 autoregulation is perturbed and TDP-43 and proteins involved in synaptic function are downregulated, leading to impairment of synaptic plasticity. Finally, we found that PPIA was downregulated in several patients with amyotrophic lateral sclerosis and amyotrophic lateral sclerosis-frontotemporal dementia, and identified a PPIA loss-of-function mutation in a patient with sporadic amyotrophic lateral sclerosis . The mutant PPIA has low stability, altered structure and impaired interaction with TDP-43. These findings strongly implicate that defective PPIA function causes TDP-43 mislocalization and dysfunction and should be considered in future therapeutic approaches.
•The cellular prion protein (PrPC) plays a dual role in prion diseases, being both a substrate for PrPSc replication, and a mediator of its toxicity.•Misfolded assemblies of several different ...pathogenic proteins may use PrPC to deliver their cytotoxic effects.•The flexible tail of PrPC may engage interactions with several different partners, including misfolded assemblies of various proteins, and execute functional as well as cytotoxic effects.•Novel pharmacological avenues for prion diseases, and potentially other neurological disorders, could be opened up by specifically targeting the N-terminus of PrPC.•The N-terminal and the C-terminal domains of PrPC appear to be functionally coupled. Thus, the globular domain of PrPC might be targeted to allosterically inhibit the detrimental activities mediated by its N-terminus.
The cellular prion protein (PrPC), a cell surface glycoprotein involved in prion disorders, has been shown to mediate the toxicity of several pathological aggregates, including its own misfolded state and some oligomeric assemblies of the amyloid β peptide, which are thought to be primarily responsible for the synaptic dysfunction characterizing Alzheimer's disease. Thus, elucidating the physiological function of PrPC, and how it could be corrupted by the interaction with misfolded proteins, may provide important insights to understand the pathological processes of prion and Alzheimer's diseases, and possibly other neurodegenerative disorders. In this manuscript, we review the data supporting a role for PrPC at the intersection of different neurodegenerative diseases, discuss potential mechanisms by which this protein could mediate neurotoxic signals, and examine therapeutic approaches that may arise from the identification of PrPC-directed compounds.
The human antigen R (HuR) is an RNA-binding protein known to modulate the expression of target mRNA coding for proteins involved in inflammation, tumorigenesis, and stress responses and is a valuable ...drug target. We previously found that dihydrotanshinone-I (DHTS, 1) prevents the association of HuR with its RNA substrate, thus imparing its function. Herein, inspired by DHTS structure, we designed and synthesized an array of ortho-quinones (tanshinone mimics) using a function-oriented synthetic approach. Among others, compound 6a and 6n turned out to be more effective than 1, showing a nanomolar K i and disrupting HuR binding to RNA in cells. A combined approach of NMR titration and molecular dynamics (MD) simulations suggests that 6a stabilizes HuR in a peculiar closed conformation, which is incompatible with RNA binding. Alpha screen and RNA-electrophoretic mobility shift assays (REMSA) data on newly synthesized compounds allowed, for the first time, the generation of structure activity relationships (SARs), thus providing a solid background for the generation of highly effective HuR disruptors.
A number of previous successful attempts in the search for therapeutics for a variety of human pathologies highlight the importance of computational technologies in the drug discovery pipeline. This ...approach, often referred to as computer-aided drug design, is unfortunately inapplicable when the precise information regarding the three-dimensional structure of disease-associated proteins or the mechanism by which they are altered to generate misfolded isoforms are missing. A typical example is represented by prion diseases, fatal pathologies of the nervous system characterized by the conformational conversion of a physiological protein called PrPC into a misfolded and infectious isoform referred to as PrPSc. Missing information regarding the atomic structure of PrPSc as well as the mechanism of templated conversion of PrPC has severely halted the discovery of effective therapies for prion diseases. In this manuscript, we review emerging opportunities to apply computer-aided techniques to target PrPC, PrPSc or to design inhibitors of prion replication, and discuss how these fast-evolving technologies could lay the groundwork for the application of entirely novel rational drug design schemes for these devastating pathologies.
The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrP
, the aggregated and infectious isoform of ...the cellular prion protein (PrP
), with largely unsuccessful results. Conversely, targeting PrP
expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrP
from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrP
from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrP
, one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrP
from the cell surface could represent a viable therapeutic strategy for prion diseases.
Prion propagation involves a templating reaction in which the infectious form of the prion protein (PrP(Sc)) binds to the cellular form (PrP(C)), generating additional molecules of PrP(Sc). While ...several regions of the PrP(C) molecule have been suggested to play a role in PrP(Sc) formation based on in vitro studies, the contribution of these regions in vivo is unclear. Here, we report that mice expressing PrP deleted for a short, polybasic region at the N terminus (residues 23-31) display a dramatically reduced susceptibility to prion infection and accumulate greatly reduced levels of PrP(Sc). These results, in combination with biochemical data, demonstrate that residues 23-31 represent a critical site on PrP(C) that binds to PrP(Sc) and is essential for efficient prion propagation. It may be possible to specifically target this region for treatment of prion diseases as well as other neurodegenerative disorders due to β-sheet-rich oligomers that bind to PrP(C).
The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc, the aggregated and infectious isoform of ...the cellular prion protein (PrPC), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti‐psychotic drug known to inhibit prion replication and toxicity by inducing the re‐localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP‐PrP to carry out a semi‐automated high content screening (HCS) of a chemical library directed at identifying non‐cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC, one of which also inhibited prion propagation and toxicity in cell cultures in a strain‐independent fashion. This study defines a new screening method and novel anti‐prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.
The absence of an effective treatment for prion diseases raises the urgency of exploring novel experimental strategies. Here, we capitalized on the rationale of removing PrPC from the plasma membrane as a way to counteract prion propagation and toxicity. To pursue this objective, we designed an experimental workflow which included a novel high‐content screening assay and employed this paradigm to challenge two chemical libraries of small molecules. We identified a new compound (HM) which acts in a prion strain‐independent fashion, directly validating the pharmacological relevance of tackling prions by removing PrPC from the cell surface.
Prion propagation involves a templating reaction in which the infectious form of the prion protein (PrPSc) binds to the cellular form (PrPC), generating additional molecules of PrPSc. While several ...regions of the PrPC molecule have been suggested to play a role in PrPSc formation based on in vitro studies, the contribution of these regions in vivo is unclear. Here, we report that mice expressing PrP deleted for a short, polybasic region at the N terminus (residues 23-31) display a dramatically reduced susceptibility to prion infection and accumulate greatly reduced levels of PrPSc. These results, in combination with biochemical data, demonstrate that residues 23-31 represent a critical site on PrPC that binds to PrPSc and is essential for efficient prion propagation. It may be possible to specifically target this region for treatment of prion diseases as well as other neurodegenerative disorders due to beta -sheet-rich oligomers that bind to PrPC.
Some mutant forms of the cellular prion protein (PrP
) carrying artificial deletions or point mutations associated with familial human prion diseases are capable of inducing spontaneous ionic ...currents across the cell membrane, conferring hypersensitivity to certain antibiotics to a wide range of cultured cells and primary cerebellar granular neurons (CGNs). These effects are abrogated when the wild type (WT) form is co-expressed, suggesting that they might be related to a physiological activity of PrP
. Interestingly, the prion protein family member Shadoo (Sho) makes cells hypersensitive to the same antibiotics as mutant PrP-s, an effect that is diminished by the co-expression of WT-PrP. Here, we report that Sho engages in another mutant PrP-like activity: it spontaneously induces large ionic currents in cultured SH-SY5Y cells, as detected by whole-cell patch clamping. These currents are also decreased by the co-expression of WT-PrP. Furthermore, deletion of the N-terminal (RXXX)
motif of Sho, mutation of the eight arginine residues of this motif to glutamines, or replacement of the hydrophobic domain by that of PrP, also diminish Sho-induced ionic currents. Our results suggest that the channel activity that is also characteristic to some pathogenic PrP mutants may be linked to a physiological function of Sho.
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