Electrophysiological Investigations of Prion Protein Roles in Health and Disease

Prion diseases are transmissible and fatal neurological disorders associated with the misfolding of cellular prion protein (PrPC) into disease-causing isoforms (PrPD) in the central nervous system. The diseases have three etiologies; acquired through exposure to the infectious PrPD, sporadic, arising from no known cause, and hereditary due to familial mutations within the PRNP gene. The manifestation of clinical signs is associated with the disruption of neuronal activity and subsequent degeneration of neurons. To generate insight into the mechanisms by which neuronal activity becomes disrupted in prion diseases, electrophysiological techniques have been applied to closely study the electrical signaling properties of neurons that lack functional PrPC as well as neurons that are developing pathological features of prion diseases due to infection or genetic mutation. In this review, we will compile the electrophysiological evidences of neurophysiological roles of PrPC, how those roles are changed in neurons that are developing prion diseases, and how disease-associated effects are exacerbated during the clinical stage of disease

Sporadic Creutzfeldt-Jakob disease infected human cerebral organoids retain the original human brain subtype features following transmission to humanized transgenic mice

Human cerebral organoids (COs) are three-dimensional self-organizing cultures of cerebral brain tissue differentiated from induced pluripotent stem cells. We have recently shown that COs are susceptible to infection with different subtypes of Creutzfeldt-Jakob disease (CJD) prions, which in humans cause different manifestations of the disease. The ability to study live human brain tissue infected with different CJD subtypes opens a wide array of possibilities from differentiating mechanisms of cell death and identifying neuronal selective vulnerabilities to testing therapeutics. However, the question remained as to whether the prions generated in the CO model truly represent those in the infecting inoculum. Mouse models expressing human prion protein are commonly used to characterize human prion disease as they reproduce many of the molecular and clinical phenotypes associated with CJD subtypes. We therefore inoculated these mice with COs that had been infected with two CJD subtypes (MV1 and MV2) to see if the original subtype characteristics (referred to as strains once transmitted into a model organism) of the infecting prions were maintained in the COs when compared with the original human brain inocula. We found that disease characteristics caused by the molecular subtype of the disease associated prion protein were similar in mice inoculated with either CO derived material or human brain material, demonstrating that the disease associated prions generated in COs shared strain characteristics with those in humans. As the first and only in vitro model of human neurodegenerative disease that can faithfully reproduce different subtypes of prion disease, these findings support the use of the CO model for investigating human prion diseases and their subtypes

Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains

Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested

Sporadic Creutzfeldt-Jakob disease prion infection of human cerebral organoids

For the transmissible, neurogenerative family of prion diseases, few human models of infection exist and none represent structured neuronal tissue. Human cerebral organoids are self-organizing, three-dimensional brain tissues that can be grown from induced pluripotent stem cells. Organoids can model aspects of neurodegeneration in Alzheimer's Disease and Down's Syndrome, reproducing tau hyperphosphorylation and amyloid plaque pathology. To determine whether organoids could be used to reproduce human prion infection and pathogenesis, we inoculated organoids with two sporadic Creutzfeldt-Jakob Disease prion subtypes. Organoids showed uptake, followed by clearance, of the infectious inoculum. Subsequent re-emergence of prion self-seeding activity indicated de novo propagation. Organoid health assays, prion titer, prion protein electrophoretic mobility and immunohistochemistry demonstrated inoculum-specific differences. Our study shows, for the first time, that cerebral organoids can model aspects of human prion disease and thus offer a powerful system for investigating different human prion subtype pathologies and testing putative therapeutics

Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors

Background Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization – homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. Results In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. Conclusion These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor internalization-induced changes in neuronal functions of the CNS

α-Synuclein seeding activity in duodenum biopsies from Parkinson's disease patients

Abnormal deposition of α-synuclein is a key feature and biomarker of Parkinson's disease. α-Synuclein aggregates can propagate themselves by a prion-like seeding-based mechanism within and between tissues and are hypothesized to move between the intestine and brain. α-Synuclein RT-QuIC seed amplification assays have detected Parkinson's-associated α-synuclein in multiple biospecimens including post-mortem colon samples. Here we show intra vitam detection of seeds in duodenum biopsies from 22/23 Parkinson's patients, but not in 6 healthy controls by RT-QuICR. In contrast, no tau seeding activity was detected in any of the biopsies. Our seed amplifications provide evidence that the upper intestine contains a form(s) of α-synuclein with self-propagating activity. The diagnostic sensitivity and specificity for PD in this biopsy panel were 95.7% and 100% respectively. End-point dilution analysis indicated up to 106 SD50 seeding units per mg of tissue with positivity in two contemporaneous biopsies from individual patients suggesting widespread distribution within the superior and descending parts of duodenum. Our detection of α-synuclein seeding activity in duodenum biopsies of Parkinson's disease patients suggests not only that such analyses may be useful in ante-mortem diagnosis, but also that the duodenum may be a source or a destination for pathological, self-propagating α-synuclein assemblies

Impacto de la formalización minera sobre el uso y defensa del territorio comunal de las CC.NN. Boca Inambari y Tres Islas, Madre de Dios

Analiza los efectos del estancamiento del proceso de formalización minera sobre las comunidades nativas Boca Inambari y Tres Islas en el departamento de Madre de Dios, Perú. Plantea que el estancamiento de la formalización minera en Madre de Dios, la incomunicación intersectorial de las entidades de decisión respecto al potencial de formalización de las Comunidades Nativas, así como las interdicciones, y los engaños de algunos consultores, han desestimulado el afán de las comunidades para ordenar sus actividades y las han conducido a asumir alternativas para subsistir. En consecuencia, identifica los efectos del estancamiento del proceso de formalización sobre el uso de otros recursos naturales diferentes al oro y sobre la soberanía territorial comunal; y caracteriza las estrategias alternativas de subsistencia de las comunidades nativas frente a las interdicciones contra la minería.Tesi

Seed amplification and neurodegeneration marker trajectories in individuals at risk of prion disease

Human prion diseases are remarkable for long incubation times followed typically by rapid clinical decline. Seed amplification assays and neurodegeneration biofluid biomarkers are remarkably useful in the clinical phase, but their potential to predict clinical onset in healthy people remains unclear. This is relevant not only to the design of preventive strategies in those at-risk of prion diseases, but more broadly, because prion-like mechanisms are thought to underpin many neurodegenerative disorders. Here, we report the accrual of a longitudinal biofluid resource in patients, controls and healthy people at risk of prion diseases, to which ultrasensitive techniques such as real-time quaking-induced conversion (RT-QuIC), and single molecule array (Simoa) digital immunoassays were applied for preclinical biomarker discovery. We studied 648 CSF and plasma samples, including 16 people who had samples taken when healthy but later developed inherited prion disease (IPD) ("converters"; range from 9.9 prior to, and 7.4 years after onset). Symptomatic IPD CSF samples were screened by RT-QuIC assay variations, before testing the entire collection of at-risk samples using the most sensitive assay. Glial fibrillary acidic protein (GFAP), neurofilament light (NfL), tau and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) levels were measured in plasma and CSF. Second generation (IQ-CSF) RT-QuIC proved 100% sensitive and specific for sporadic Creutzfeldt-Jakob disease (sCJD), iatrogenic (iCJD) and familial CJD phenotypes, and subsequently detected seeding activity in four presymptomatic CSF samples from three E200K carriers; one converted in under two months while two remain asymptomatic after at least three years' follow-up. A bespoke HuPrP P102L RT-QuIC showed partial sensitivity for P102L disease. No compatible RT-QuIC assay was discovered for classical 6-OPRI, A117V and D178N, and these at-risk samples tested negative with bank vole RT-QuIC. Plasma GFAP and NfL, and CSF NfL levels emerged as proximity markers of neurodegeneration in the typically slow IPDs (e.g. P102L), with significant differences in mean values segregating healthy control from IPD carriers (within 2 years to onset) and symptomatic IPD cohorts; plasma GFAP appears to change before NfL, and before clinical conversion. In conclusion, we show distinct biomarker trajectories in fast and slow IPDs. Specifically, we identify several years of presymptomatic seeding positivity in E200K, a new proximity marker (plasma GFAP) and sequential neurodegenerative marker evolution (plasma GFAP followed by NfL) in slow IPDs. We suggest a new preclinical staging system featuring clinical, seeding and neurodegeneration aspects, for validation with larger prion at-risk cohorts, and with potential application to other neurodegenerative proteopathies

Efficacy of Wex-cide 128 disinfectant against multiple prion strains

Prion diseases are transmissible, fatal neurologic diseases that include Creutzfeldt-Jakob Disease (CJD) in humans, chronic wasting disease (CWD) in cervids, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. Prions are extremely difficult to inactivate and established methods to reduce prion infectivity are often dangerous, caustic, expensive, or impractical. Identifying viable and safe methods for treating prion contaminated materials is important for hospitals, research facilities, biologists, hunters, and meat-processors. For three decades, some prion researchers have used a phenolic product called Environ LpH (eLpH) to inactivate prions. ELpH has been discontinued, but a similar product, Wex-cide 128, containing the similar phenolic chemicals as eLpH is now available. In the current study, we directly compared the anti-prion efficacy of eLpH and Wex-cide 128 against prions from four different species (hamster 263K, cervid CWD, mouse 22L and human CJD). Decontamination was performed on either prion infected brain homogenates or prion contaminated steel wires and mouse bioassay was used to quantify the remaining prion infectivity. Our data show that both eLpH and Wex-cide 128 removed 4.0–5.5 logs of prion infectivity from 22L, CWD and 263K prion homogenates, but only about 1.25–1.50 logs of prion infectivity from human sporadic CJD. Wex-cide 128 is a viable substitute for inactivation of most prions from most species, but the resistance of CJD to phenolic inactivation is a concern and emphasizes the fact that inactivation methods should be confirmed for each target prion strain

Roles of the SH2 and SH3 domains in the regulation of neuronal Src kinase functions

Previous studies demonstrated that intra-domain interactions between Src family kinases (SFKs), stabilized by binding of the phosphorylated C-terminus to the SH2 domain and/or binding of the SH2 kinase linker to the SH3 domain, lock the molecules in a closed conformation, disrupt the kinase active site, and inactivate SFKs. Here we report that the up-regulation of N-methyl-d-aspartate receptors (NMDARs) induced by expression of constitutively active neuronal Src (n-Src), in which the C-terminus tyrosine is mutated to phenylalanine (n-Src/Y535F), is significantly reduced by dysfunctions of the SH2 and/or SH3 domains of the protein. Furthermore, we found that dysfunctions of SH2 and/or SH3 domains reduce auto-phosphorylation of the kinase activation loop, depress kinase activity, and decrease NMDAR phosphorylation. The SH2 domain plays a greater regulatory role than the SH3 domain. Our data also show that n-Src binds directly to the C-terminus of the NMDAR NR2A subunit in vitro, with a K D of 108.2 ± 13.3 nm. This binding is not Src kinase activity-dependent, and dysfunctions of the SH2 and/or SH3 domains do not significantly affect the binding. These data indicate that the SH2 and SH3 domains may function to promote the catalytic activity of active n-Src, which is important in the regulation of NMDAR functions. © 2010 The Authors Journal compilation © 2010 FEBS