Sensitive detection of pathological seeds of α-synuclein, tau and prion protein on solid surfaces.

Prions or prion-like aggregates such as those composed of PrP, α-synuclein, and tau are key features of proteinopathies such as prion, Parkinson's and Alzheimer's diseases, respectively. Their presence on solid surfaces may be biohazardous under some circumstances. PrP prions bound to solids are detectable by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays if the solids can be immersed in assay wells or the prions transferred to pads. Here we show that prion-like seeds can remain detectable on steel wires for at least a year, or even after enzymatic cleaning and sterilization. We also show that contamination of larger objects with pathological seeds of α-synuclein, tau, and PrP can be detected by simply assaying a sampling medium that has been transiently applied to the surface. Human α-synuclein seeds in dementia with Lewy bodies brain tissue were detected by α-synuclein RT-QuIC after drying of tissue dilutions with concentrations as low as 10-6 onto stainless steel. Tau RT-QuIC detected tau seeding activity on steel exposed to Alzheimer's disease brain tissue diluted as much as a billion fold. Prion RT-QuIC assays detected seeding activity on plates exposed to brain dilutions as extreme as 10-5-10-8 from prion-affected humans, sheep, cattle and cervids. Sampling medium collected from surgical instruments used in necropsies of sporadic Creutzfeldt-Jakob disease-infected transgenic mice was positive down to 10-6 dilution. Sensitivity for prion detection was not sacrificed by omitting the recombinant PrP substrate from the sampling medium during its application to a surface and subsequent storage as long as the substrate was added prior to performing the assay reaction. Our findings demonstrate practical prototypic surface RT-QuIC protocols for the highly sensitive detection of pathologic seeds of α-synuclein, tau, and PrP on solid objects.

Influence of CReatine supplementation on mUScle mass and strength after stroke (ICaRUS Stroke Trial): study protocol for a randomized controlled trial.

BACKGROUND: Stroke is a leading cause of mortality and disability, and its sequelae are associated with inadequate food intake which can lead to sarcopenia. The aim of this study is to verify the effectiveness of creatine supplementation on functional capacity, strength, and changes in muscle mass during hospitalization for stroke compared to usual care. An exploratory subanalysis will be performed to assess the inflammatory profiles of all participants, in addition to a follow-up 90 days after stroke, to verify functional capacity, muscle strength, mortality, and quality of life. METHODS: Randomized, double-blind, unicenter, parallel-group trial including individuals with ischemic stroke in the acute phase. The duration of the trial for the individual subject will be approximately 90 days, and each subject will attend a maximum of three visits. Clinical, biochemical, anthropometric, body composition, muscle strength, functional capacity, degree of dependence, and quality of life assessments will be performed. Thirty participants will be divided into two groups: intervention (patients will intake one sachet containing 10g of creatine twice a day) and control (patients will intake one sachet containing 10g of placebo [maltodextrin] twice a day). Both groups will receive supplementation with powdered milk protein serum isolate to achieve the goal of 1.5g of protein/kg of body weight/day and daily physiotherapy according to the current rehabilitation guidelines for patients with stroke. Supplementation will be offered during the 7-day hospitalization. The primary outcomes will be functional capacity, strength, and changes in muscle mass after the intervention as assessed by the Modified Rankin Scale, Timed Up and Go test, handgrip strength, 30-s chair stand test, muscle ultrasonography, electrical bioimpedance, and identification of muscle degradation markers by D3-methylhistidine. Follow-up will be performed 90 days after stroke to verify functional capacity, muscle strength, mortality, and quality of life. DISCUSSION: The older population has specific nutrient needs, especially for muscle mass and function maintenance. Considering that stroke is a potentially disabling event that can lead the affected individual to present with numerous sequelae, it is crucial to study the mechanisms of muscle mass loss and understand how adequate supplementation can help these patients to better recover. TRIAL REGISTRATION: The Brazilian Clinical Trials Registry (ReBEC) RBR-9q7gg4 . Registered on 21 January 2019.

Neuronal excitatory-to-inhibitory balance is altered in cerebral organoid models of genetic neurological diseases.

The neuro-physiological properties of individuals with genetic pre-disposition to neurological disorders are largely unknown. Here we aimed to explore these properties using cerebral organoids (COs) derived from fibroblasts of individuals with confirmed genetic mutations including PRNPE200K, trisomy 21 (T21), and LRRK2G2019S, which are associated with Creutzfeldt Jakob disease, Down Syndrome, and Parkinson's disease. We utilized no known disease/healthy COs (HC) as normal function controls. At 3-4 and 6-10 months post-differentiation, COs with mutations showed no evidence of disease-related pathology. Electrophysiology assessment showed that all COs exhibited mature neuronal firing at 6-10 months old. At this age, we observed significant changes in the electrophysiology of the COs with disease-associated mutations (dCOs) as compared with the HC, including reduced neuronal network communication, slowing neuronal oscillations, and increased coupling of delta and theta phases to the amplitudes of gamma oscillations. Such changes were linked with the detection of hypersynchronous events like spike-and-wave discharges. These dysfunctions were associated with altered production and release of neurotransmitters, compromised activity of excitatory ionotropic receptors including receptors of kainate, AMPA, and NMDA, and changed levels and function of excitatory glutamatergic synapses and inhibitory GABAergic synapses. Neuronal properties that modulate GABAergic inhibition including the activity of Na-K-Cl cotransport 1 (NKCC1) in Cl- homeostasis and the levels of synaptic and extra-synaptic localization of GABA receptors (GABARs) were altered in the T21 COs only. The neurosteroid allopregnanolone, a positive modulator of GABARs, was downregulated in all the dCOs. Treatment with this neurosteroid significantly improved the neuronal communication in the dCOs, possibly through improving the GABAergic inhibition. Overall, without the manifestation of any disease-related pathology, the genetic mutations PRNPE200K, T21, and LRRK2G2019S significantly altered the neuronal network communication in dCOs by disrupting the excitatory-to-inhibitory balance.

Detection of chronic wasting disease in mule and white-tailed deer by RT-QuIC analysis of outer ear.

Efforts to contain the spread of chronic wasting disease (CWD), a fatal, contagious prion disease of cervids, would be aided by the availability of additional diagnostic tools. RT-QuIC assays allow ultrasensitive detection of prion seeds in a wide variety of cervid tissues, fluids and excreta. The best documented antemortem diagnostic test involving RT-QuIC analysis targets lymphoid tissue in rectal biopsies. Here we have tested a more easily accessed specimen, ear pinna punches, using an improved RT-QuIC assay involving iron oxide magnetic extraction to detect CWD infections in asymptomatic mule and white-tailed deer. Comparison of multiple parts of the ear pinna indicated that a central punch spanning the auricular nerve provided the most consistent detection of CWD infection. When compared to results obtained from gold-standard retropharyngeal lymph node specimens, our RT-QuIC analyses of ear samples provided apparent diagnostic sensitivity (81%) and specificity (91%) that rivaled, or improved upon, those observed in previous analyses of rectal biopsies using RT-QuIC. These results provide evidence that RT-QuIC analysis of ear pinna punches may be a useful approach to detecting CWD infections in cervids.

Human cerebral organoids as a therapeutic drug screening model for Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob Disease (CJD) is a fatal, currently incurable, neurodegenerative disease. The search for candidate treatments would be greatly facilitated by the availability of human cell-based models of prion disease. Recently, an induced pluripotent stem cell derived human cerebral organoid model was shown to take up and propagate human CJD prions. This model offers new opportunities to screen drug candidates for the treatment of human prion diseases in an entirely human genetic background. Here we provide the first evidence that human cerebral organoids can be a viable model for CJD drug screening by using an established anti-prion compound, pentosan polysulfate (PPS). PPS delayed prion propagation in a prophylactic-like treatment paradigm and also alleviated propagation when applied following establishment of infection in a therapeutic-like treatment paradigm. This study demonstrates the utility of cerebral organoids as the first human 3D cell culture system for screening therapeutic drug candidates for human prion diseases.

RNA modulates aggregation of the recombinant mammalian prion protein by direct interaction.

Recent studies have proposed that nucleic acids act as potential cofactors for protein aggregation and prionogenesis. By means of sedimentation, transmission electron microscopy, circular dichroism, static and dynamic light scattering, we have studied how RNA can influence the aggregation of the murine recombinant prion protein (rPrP). We find that RNA, independent of its sequence, source and size, modulates rPrP aggregation in a bimodal fashion, affecting both the extent and the rate of rPrP aggregation in a concentration dependent manner. Analogous to RNA-induced liquid-liquid phase transitions observed for other proteins implicated in neurodegenerative diseases, high protein to RNA ratios stimulate rPrP aggregation, while low ratios suppress it. However, the latter scenario also promotes formation of soluble oligomeric aggregates capable of seeding de novo rPrP aggregation. Furthermore, RNA co-aggregates with rPrP and thereby gains partial protection from RNase digestion. Our results also indicate that rPrP interacts with the RNAs with its N-terminus. In summary, this study elucidates the proposed adjuvant role of RNA in prion protein aggregation and propagation, and thus advocates an auxiliary role of the nucleic acids in protein aggregation in general.

Transfer of mupirocin resistance from Staphylococcus haemolyticus clinical strains to Staphylococcus aureus through conjugative and mobilizable plasmids.

Coagulase-negative staphylococci are thought to act as reservoirs of antibiotic resistance genes that can be transferred to Staphylococcus aureus, thus hindering the combat of this bacterium. In this work, we analyzed the presence of plasmids conferring resistance to the antibiotic mupirocin-widely used to treat and prevent S. aureus infections in hospital environments-in nosocomial S. haemolyticus strains. About 12% of the 75 strains tested were resistant to mupirocin, and this phenotype was correlated with the presence of plasmids. These plasmids were shown to be diverse, being either conjugative or mobilizable, and capable of transferring mupirocin resistance to S. aureus Our findings reinforce that S. haemolyticus, historically and mistakenly considered as a less important pathogen, is a reservoir of resistance genes which can be transferred to other bacteria, such as S. aureus, emphasizing the necessity of more effective strategies to detect and combat this emergent opportunistic pathogen.

Toxicological Evaluation of Anti-Scrapie Trimethoxychalcones and Oxadiazoles.

An altered form of the cellular prion protein, the PrPScor PrPRes, is implicated in the occurrence of the still untreatable transmissible spongiform encephalopathies. We have previously synthesized and characterized aromatic compounds that inhibit protease-resistant prion protein (PrPRes) accumulation in scrapie-infected cells. These compounds belong to different chemical classes, including acylhydrazones, chalcones and oxadiazoles. Some of the active compounds were non-toxic to neuroblastoma cells in culture and seem to possess drugable properties, since they are in agreement with the Lipinski´s rule of 5 and present desirable pharmacokinetic profiles as predicted in silico. Before the evaluation of the in vivo efficacy of the aromatic compounds in scrapie-infected mice, safety assessment in healthy mice is needed. Here we used Swiss mice to evaluate the acute toxicity profile of the six most promising anti-prionic compounds, the 2,4,5-trimethoxychalcones (J1, J8, J20 and J35) and the 1,3,4-oxadiazoles (Y13 and Y17). One single oral administration (300 mg/kg) of J1, J8, J20, J35, Y13 and Y17 or repeated intraperitoneal administration (10 mg/kg, 3 times a week, for 4 weeks) of J1, J8 and J35, did not elicit toxicity in mice. We strongly believe that the investigated trimethoxychalcones and oxadiazoles are interesting compounds to be further analyzed in vivo against prion diseases.

New approaches for the selection and evaluation of anti-prion organic compounds.

Transmissible spongiform encephalopathies (TSEs) are infectious neurodegenerative disorders for which symptomatic, curative, or prophylactic treatments are not available. TSEs arise as a consequence of the conversion of soluble cellular prion protein (PrP(C)) into the scrapie isoform (PrP(Sc)), which aggregates and accumulates in the central nervous system. Proposed drugs against TSEs range from small organic compounds to antibodies; various therapeutic strategies have been proposed, including blocking the conversion of PrP(C) to PrP(Sc), increasing PrP(Sc) clearance, and/or stabilizing PrP(C). While several compounds have been effective in vitro and in animal models, none have proven effective in clinical studies to date. Such lack of in vivo efficacy is attributable to high compound toxicity and the lack of permeability of the selected compounds across the blood-brain barrier. In this review, we discuss recent advances in the screening and evaluation of organic compounds for anti-prion activity using multiple approaches, including initial screening in prion-infected cell cultures, in silico prediction of pharmacokinetic and physicochemical properties, ex vivo evaluation of cellular toxicity, and in vitro assays using purified recombinant prion proteins. The main challenges for effective discrimination of candidate lead compounds as therapeutic agents for TSEs, and the disadvantages of each screening strategy are discussed. We propose that a combination of in vitro, ex vivo, and in silico approaches would be useful for the rapid identification of novel anti-prion drug candidates with suitable pharmacokinetic and pharmacodynamic properties that would support their use as drugs.

Anti-prion activity of a panel of aromatic chemical compounds: in vitro and in silico approaches.

The prion protein (PrP) is implicated in the Transmissible Spongiform Encephalopathies (TSEs), which comprise a group of fatal neurodegenerative diseases affecting humans and other mammals. Conversion of cellular PrP (PrP(C)) into the scrapie form (PrP(Sc)) is the hallmark of TSEs. Once formed, PrP(Sc) aggregates and catalyzes PrP(C) misfolding into new PrP(Sc) molecules. Although many compounds have been shown to inhibit the conversion process, so far there is no effective therapy for TSEs. Besides, most of the previously evaluated compounds failed in vivo due to poor pharmacokinetic profiles. In this work we propose a combined in vitro/in silico approach to screen for active anti-prion compounds presenting acceptable drugability and pharmacokinetic parameters. A diverse panel of aromatic compounds was screened in neuroblastoma cells persistently infected with PrP(Sc) (ScN2a) for their ability to inhibit PK-resistant PrP (PrP(Res)) accumulation. From ∼200 compounds, 47 were effective in decreasing the accumulation of PrP(Res) in ScN2a cells. Pharmacokinetic and physicochemical properties were predicted in silico, allowing us to obtain estimates of relative blood brain barrier permeation and mutagenicity. MTT reduction assays showed that most of the active compounds were non cytotoxic. Compounds that cleared PrP(Res) from ScN2a cells, were non-toxic in the MTT assay, and presented a good pharmacokinetic profile were investigated for their ability to inhibit aggregation of an amyloidogenic PrP peptide fragment (PrP(109-149)). Molecular docking results provided structural models and binding affinities for the interaction between PrP and the most promising compounds. In summary, using this combined in vitro/in silico approach we have identified new small organic anti-scrapie compounds that decrease the accumulation of PrP(Res) in ScN2a cells, inhibit the aggregation of a PrP peptide, and possess pharmacokinetic characteristics that support their drugability. These compounds are attractive candidates for prion disease therapy.