Prions, transmissible agents that cause Creutzfeldt-Jakob disease (CJD) and other prion diseases, are known to resist conventional sterilization procedures. Iatrogenic transmission of classical CJD via neurosurgical instruments is well documented and the involvement of lymphoreticular tissues in variant CJD (vCJD), together with the unknown population prevalence of asymptomatic vCJD infection, has led to concerns about transmission from a wide range of surgical procedures. To address this problem, conditions were sought that destroy PrP Sc from vCJD-infected human tissue and eradicate RML prion infectivity adsorbed onto surgical steel. Seven proteolytic enzymes were evaluated individually and in pairs at a range of temperatures and pH values and the additional effects of detergents, lipases and metal ions were assessed. A combination of proteinase K and Pronase, in conjunction with SDS, was shown to degrade PrP Sc material from highly concentrated vCJD-infected brain preparations to a level below detection. When RML prion-infected wires were exposed to the same enzymic treatment, intracerebral bioassay in highly susceptible hosts showed virtually no infectivity. The prion-degrading reagents identified in this study are readily available, inexpensive, non-corrosive to instruments, non-hazardous to staff and compatible with current equipment and procedures used in hospital sterilization units.

There are two common forms of prion protein (PrP) in humans, with either methionine or valine at position 129. This polymorphism is a powerful determinant of the genetic susceptibility of humans toward both sporadic and acquired forms of prion disease and restricts propagation of particular prion strains. Despite its key role, we have no information on the effect of this mutation on the structure, stability, folding, and dynamics of the cellular form of PrP (PrP C ). Here, we show that the mutation has no measurable effect on the folding, dynamics, and stability of PrP C . Our data indicate that the 129M/V polymorphism does not affect prion propagation through its effect on PrP C ; rather, its influence is likely to be downstream in the disease mechanism. We infer that the M/V effect is mediated through the conformation or stability of disease-related PrP (PrP Sc ) or intermediates or on the kinetics of their formation.The prion diseases are a group of fatal neurodegenerative diseases that include scrapie in sheep and goats; bovine spongiform encephalopathy (BSE) 1 in cattle; and Creutzfeldt-Jakob disease (CJD), Gerstmann-Strä ussler-Scheinker disease, fatal familial insomnia (FFI), and kuru in humans. The human diseases may be inherited, arise sporadically, or be acquired through exposure to infectious prions (1, 2). Although rare in humans, intense interest has focused on these diseases both because of their unique biology and because of the occurrence of variant CJD, a new form of human prion disease, and the experimental evidence that it is caused by a BSE-like prion strain (3-5).According to the "protein-only" hypothesis (6), prions are composed principally or entirely of abnormal isoforms of hostencoded prion protein (PrP) (7). The disease-related isoform, PrP Sc , is derived from its normal cellular precursor, PrP C , by a post-translational process that involves conformational change. PrP Sc can be distinguished biochemically from PrP C by its partial protease resistance and detergent insolubility.Although the precise molecular events involved in this conversion remain ill defined, molecular genetic and in vitro studies support the hypothesis that some sort of direct interaction between PrP Sc and either PrP C or some less organized state occurs. This interaction results in the PrP Sc conformation being imposed upon the substrate protein, and the process of conversion is favored by sequence complementarity (8 -13). A key piece of evidence supporting this and the protein-only hypothesis in general is the finding that the large majority of cases of sporadic CJD are homozygous with respect to a common polymorphism at position 129 in the human prion protein, in which either methionine or valine can be encoded (only ϳ49% of the UK population are homozygous with respect to this polymorphism) (9).Elderly survivors of the kuru epidemic (an acquired prion disease largely restricted to the Fore linguistic group of the Papua New Guinea Highlands, which was transmitted during endocannibalistic feasts) who had mult...

Due to their ability to inhibit antigeninduced T-cell activation in vitro and in vivo, anergic T cells can be considered part of the spectrum of immunoregulatory T lymphocytes. Here we report that both murine and human anergic T cells can impair the ability of parenchymal cells (including endothelial and epithelial cells) to establish cell-cell interactions necessary to sustain leukocyte migration in vitro and tissue infiltration in vivo. The inhibition is reversible and cell-contact dependent but does not require cognate recognition of the parenchymal cells to occur. Instrumental to this effect is the increased cell surface expression and enzymatic activity of molecules such as CD26 (dipeptidyl-peptidase IV), which may act by metabolizing chemoattractants bound to the endothelial/epithelial cell surface. These results describe a previously unknown antigen-independent antiinflammatory activity by locally generated anergic T cells and define a novel mechanism for the long-known immunoregulatory properties of these cells. IntroductionT-cell anergy has been defined as a "cellular state in which a lymphocyte is alive but fails to display certain functional responses (including cell division and interleukin 2 [IL-2] production) when optimally stimulated through both its antigen-specific receptor and any other receptor that is normally required for full activation." 1 Anergic T cells can be generated in vitro and in vivo by various mechanisms, 1,2 all involving partial or inappropriate stimulation. While losing their proliferative and effector potential, anergic T cells have long been known to be able to exert immunoregulation. The potential for anergic T cells to act as suppressor cells came first from a superantigen in vivo model in which anergic T cells acted as efficient suppressor cells in an antigen-nonspecific manner. 3 More recently, murine anergic T cells either generated in vivo or rendered anergic in vitro with immobilized anti-CD3 and adoptively transferred have been shown to prolong skin allograft survival in an antigen-specific manner. 4,5 In the human system, anergic CD4 ϩ T cells were shown to exert contact-dependent and antigen-specific suppression in vitro. 6,7 The mechanism by which anergic T cells exert their immunoregulatory properties appears to be indirect by altering the antigen presenting cells (APCs) immunogenicity 8,9 in a cell-cell contactdependent manner. The molecular basis for this effect is still unknown and it has been hypothesized to involve the induction of "regulatory" molecules on the anergic T cells, capable of delivering negative signals to the APC. 2 It has recently become clear that the initial stages of T-cell activation are mediated by antigen-independent interactions, which establish areas of focal contact between T cells and APCs. 10,11 Such interactions are initiated by chemoattractant-induced cell polarization and subsequent redistribution of adhesion molecules on the T-cell surface. These, in turn, allow T-cell receptor (TCR) interactions with the major histocompatibili...

Cellular prion protein (PrP) is prominently expressed in brain, in differentiated neurons but also in neural stem/precursor cells (NPCs). The misfolding of PrP is a central event in prion diseases, yet the physiological function of PrP is insufficiently understood. Although PrP has been reported to associate with the neural cell adhesion molecule (NCAM), the consequences of concerted PrP-NCAM action in NPC physiology are unknown. Here, we generated NPCs from the subventricular zone (SVZ) of postnatal day 5 wild-type and PrP null (2/2) mice and observed that PrP is essential for proper NPC proliferation and neuronal differentiation. Moreover, we found that PrP is required for the NPC response to NCAM-induced neuronal differentiation. In the absence of PrP, NCAM not only fails to promote neuronal differentiation but also induces an accumulation of doublecortin-positive neuronal progenitors at the proliferation stage. In agreement, we noted an increase in cycling neuronal progenitors in the SVZ of PrP2/2 mice compared with PrP1/1 mice, as evidenced by double labeling for the proliferation marker Ki67 and doublecortin as well as by 5-bromo-2 0 -deoxyuridine incorporation experiments. Additionally, fewer newly born neurons were detected in the rostral migratory stream of PrP2/2 mice. Analysis of the migration of SVZ cells in microexplant cultures from wild-type and PrP2/2 mice revealed no differences between genotypes or a role for NCAM in this process. Our data demonstrate that PrP plays a critical role in neuronal differentiation of NPCs and suggest that this function is, at least in part, NCAM-dependent.

Identification of the unique features of human brain development and function can be critical towards the elucidation of intricate processes such as higher cognitive functions and human-specific pathologies like neuropsychiatric and behavioral disorders. The developing primate and human central nervous system (CNS) are distinguished by expanded progenitor zones and a protracted time course of neurogenesis, leading to the expansion in brain size, prominent gyral anatomy, distinctive synaptic properties, and complex neural circuits. Comparative genomic studies have revealed that adaptations of brain capacities may be partly explained by human-specific genetic changes that impact the function of proteins associated with neocortical expansion, synaptic function, and language development. However, the formation of complex gene networks may be most relevant for brain evolution. Indeed, recent studies identified distinct human-specific gene expression patterns across developmental time occurring in brain regions linked to cognition. Interestingly, such modules show speciesspecific divergence and are enriched in genes associated with neuronal development and synapse formation whilst also being implicated in neuropsychiatric diseases. microRNAs represent a powerful component of gene-regulatory networks by promoting spatiotemporal post-transcriptional control of gene expression in the human and primate brain. It has also been suggested that the divergence in miRNA expression plays an important role in shaping gene expression divergence among species. Primatespecific and human-specific miRNAs are principally involved in progenitor proliferation and neurogenic processes but also associate with human cognition, and neurological disorders. Human embryonic or induced pluripotent stem cells and brain organoids, permitting experimental access to neural cells and differentiation stages that are otherwise difficult or impossible to reach in humans, are an essential means for studying species-specific brain miRNAs. Single-cell sequencing approaches can further decode refined miRNA-mRNA interactions during developmental transitions. Elucidating species-specific miRNA regulation will shed new light into the mechanisms that control spatiotemporal events during human brain development and disease, an important step towards fostering novel, holistic and effective therapeutic approaches for neural disorders. In this review, we discuss species-specific regulation of miRNA function, its contribution to the evolving features of the human brain and in neurological disease, with respect also to future therapeutic approaches.

Parkinson’s disease (PD) is a common progressive neurodegenerative disorder characterized by loss of striatal-projecting dopaminergic neurons of the ventral forebrain, resulting in motor and cognitive deficits. Despite extensive efforts in understanding PD pathogenesis, no disease-modifying drugs exist. Recent advances in cell reprogramming technologies have facilitated the generation of patient-derived models for sporadic or familial PD and the identification of early, potentially triggering, pathological phenotypes while they provide amenable systems for drug discovery. Emerging developments highlight the enhanced potential of using more sophisticated cellular systems, including neuronal and glial co-cultures as well as three-dimensional systems that better simulate the human pathophysiology. In combination with high-throughput high-content screening technologies, these approaches open new perspectives for the identification of disease-modifying compounds. In this review, we discuss current advances and the challenges ahead in the use of patient-derived induced pluripotent stem cells for drug discovery in PD. We address new concepts implicating non-neuronal cells in disease pathogenesis and highlight the necessity for functional assays, such as calcium imaging and multi-electrode array recordings, to predict drug efficacy. Finally, we argue that artificial intelligence technologies will be pivotal for analysis of the large and complex data sets obtained, becoming game-changers in the process of drug discovery.

According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrP(Sc), (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrP(Sc) or on prion infectivity.

Integrating differential RNA and miRNA expression during neuronal lineage induction of human embryonic stem cells we identified miR-934, a primate-specific miRNA that displays a stage-specific expression pattern during progenitor expansion and early neuron generation. We demonstrate the biological relevance of this finding by comparison with data from early to mid-gestation human cortical tissue. Further we find that miR-934 directly controls progenitor to neuroblast transition and impacts on neurite growth of newborn neurons. In agreement, miR-934 targets are involved in progenitor proliferation and neuronal differentiation whilst miR-934 inhibition results in profound global transcriptome changes associated with neurogenesis, axonogenesis, neuronal migration and neurotransmission. Interestingly, miR-934 inhibition affects the expression of genes associated with the subplate zone, a transient compartment most prominent in primates that emerges during early corticogenesis. Our data suggest that mir-934 is a novel regulator of early human neurogenesis with potential implications for a species-specific evolutionary role in brain function.