Conditions of endoplasmic reticulum stress favor the accumulation of cytosolic prion protein.

After signal sequence-dependent targeting to the endoplasmic reticulum (ER), prion protein (PrP) undergoes several post-translational modifications, including glycosylation, disulfide bond formation, and the addition of a glycosylphosphatidylinositol anchor. As a result, multiple isoforms are generated. Because of the intrinsic weakness of the PrP signal sequence, a fraction of newly synthesized molecules fails to translocate and localizes to the cytosol. The physiopathologic role of this cytosolic isoform is still being debated. Here we have shown that, in both cultured cell lines and primary neurons, ER stress conditions weaken PrP co-translational translocation, favoring accumulation of aggregation-prone cytosolic species, which retain the signal sequence but lack N-glycans and disulfides. Inhibition of proteasomes further increases the levels of cytosolic PrP. Overexpression of spliced XBP1 facilitates ER translocation, suggesting that downstream elements of the Ire1-XBP1 pathway are involved in PrP targeting. These studies reveal a link between ER stress and the formation of cytosolic PrP isoforms potentially endowed with novel signaling or cytotoxic functions

Liquid-Liquid Phase Separation in Physiology and Pathophysiology of the Nervous System

Molecules within cells are segregated into functional domains to form various organelles. While some of those organelles are delimited by lipid membranes demarcating their constituents, others lack a membrane enclosure. Recently, liquid-liquid phase separation (LLPS) revolutionized our view of how segregation of macromolecules can produce membraneless organelles. While the concept of LLPS has been well studied in the areas of soft matter physics and polymer chemistry, its significance has only recently been recognized in the field of biology. It occurs typically between macromolecules that have multivalent interactions. Interestingly, these features are present in many molecules that exert key functions within neurons. In this review, we cover recent topics of LLPS in different contexts of neuronal physiology and pathology

Cytosolic Prion Protein (PrP) Is Not Toxic in N2a Cells and Primary Neurons Expressing Pathogenic PrP Mutations

Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases

Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation

The cellular mechanisms by which prions cause neurological dysfunction are poorly understood. To address this issue, we have been using cultured cells to analyze the localization, biosynthesis, and metabolism of PrP molecules carrying mutations associated with familial prion diseases. We report here that mutant PrP molecules are delayed in their maturation to an endoglycosidase H-resistant form after biosynthetic labeling, suggesting that they are impaired in their exit from the endoplasmic reticulum (ER). However, we find that proteasome inhibitors have no effect on the maturation or turnover of either mutant or wild-type PrP molecules. Thus, in contrast to recent studies from other laboratories, our work indicates that PrP is not subject to retrotranslocation from the ER into the cytoplasm prior to degradation by the proteasome. We find that in transfected cells, but not in cultured neurons, proteasome inhibitors cause accumulation of an unglycosylated, signal peptide-bearing form of PrP on the cytoplasmic face of the ER membrane. Thus, under conditions of elevated expression, a small fraction of PrP chains is not translocated into the ER lumen during synthesis, and is rapidly degraded in the cytoplasm by the proteasome. Finally, we report a previously unappreciated artifact caused by treatment of cells with proteasome inhibitors: an increase in PrP mRNA level and synthetic rate when the protein is expressed from a vector containing a viral promoter. We suggest that this phenomenon may explain some of the dramatic effects of proteasome inhibitors observed in other studies. Our results clarify the role of the proteasome in the cell biology of PrP, and suggest reasonable hypotheses for the molecular pathology of inherited prion diseases

Characterization of historical coating mortars of La Ceramo factory in Valencia

[EN] The objective of this paper was to characterize historical coating mortars taken from the La Ceramo factory, in Valencia, by means of historical, in situ and sample collection at various points in the building, for subsequent laboratory tests. The physical-mechanical characteristics studied were: compressive strength, water content, granulometry by sieving method, identification of calcium carbonate with hydrochloric acid, surface hardness, water absorption, apparent porosity and bulk density. The results showed that the mortars composed of cement and lime collected did not present very positive characteristics in the aspects analyzed in this study, resulting in material with low quality, both in its initial composition and in function of the external influences suffered over time. Regarding the other areas of lime mortar, these presented better results, although less resistant than those of cement, were shown to have good quality. It can also be observed that lime mortars, even having a similar composition in their origin, when applied at different points of the factory acquired uneven characteristics over time, directly related to the local conditions of the coated walls. Finally, the need for preventive conservation in buildings of historical interest makes this paper of investigative and scientific nature, since the knowledge of the original materials is the initial step to a good intervention and not to accelerate the process of degradation of historical constructions.To FAPESP – Fundação de Amparo a Pesquisa do Estado de São Paulo, for the granting of scholarships in the BEPE – Internship Research Internship Grant the first two authors.Sutti, ML.; Aguiar, MOSD.; Fioriti, CF.; Christófani, MPH. (2019). Characterization of historical coating mortars of La Ceramo factory in Valencia. VITRUVIO - International Journal of Architectural Technology and Sustainability. 4(1):58-73. https://doi.org/10.4995/vitruvio-ijats.2019.11485SWORD587341Asociación Española de Normalización - UNE (2012). EN 9331-1: Ensayos para determinar las propriedades geométricas de los áridos, España.Asociación Española de Normalización - UNE (2014). EN 13279-2: Yesos de construcción y conglomerantes a base de yeso para la construcción, España.Associação Brasileira de Normas Técnicas - ABNT (2009). NBR 9778: Argamassa e concreto endurecidos - determinação da absorção de água, índice de vazios e massa específica, Brasil.Calia, M. (2017). CReV - Universitat Politècnica de Valencia, España.Falcão, S. (2010). Caracterização das argamassas antigas da igreja do Colégio de Portimão. Dissertação (Mestrado) - Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal.Lanzinha, J. C. G. (1998). Propriedades higrotérmicas de materiais de construção. Dissertação (Mestrado) - Faculdade de Ciências e Tecnologia da Universidade de Coimbra, Coimbra, Portugal.Pastor, R. C.; Agórriz, E. A. (2009). Apuntes de conglomerantes y materiales componentes de morteros y hormigones - Universidad Politecnica de Valencia - Departamento de Constucciones Arquitectónicas, Valencia, España.Plagiuca, A. (2017). CReV - Conoscenza Recupero Valorizzazione - Università degli Studi dela Basilicat, Potenza, Italy.Rodrigues, P. N. (2013). Caracterização das argamassas históricas da ruína de São Miguel Arcanjo/RS. Dissertação (Mestrado) - Universidade Federal de Santa Maria - Centro de Tecnologia, Santa Maria, Brasil.Santos, P. S. (1989). Ciência e tecnologia de argilas, Vol. 1, Edgard Blucher Ltda., São Paulo, Brasil.Santos, A. S. (2003). Nova abordagem na caracterização de argamassas antigas. Atas do 3º ENCORE - Encontro Sobre Conservação e Reabilitação de Edifícios, LNEC, Lisboa, Portugal.Válek, J.; Veiga, R. (2005). Characterization of mechanical properties of historic mortars - testing of irregular samples. Proceedings of 9th International Conference on Structural Studies, Repairs and Maintenance of Heritage Architecture (STREMAH 2005), University of Malta, Malta.Velosa, A. (2006). Argamassas de cal com pozolanas para revestimento de paredes antigas. Tese (Doutorado) - Universidade de Aveiro, Aveiro, Portugal

Cell Type-Specific Neuroprotective Activity of Untranslocated Prion Protein

Background: A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP). However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions. Principal Findings: Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells. Significance: These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function

The role of abnormal forms of the prion protein in the pathogenesis of inherited prion diseases

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Essential Role of Coiled Coils for Aggregation and Activity of Q/N-Rich Prions and PolyQ Proteins

SummaryThe functional switch of glutamine/asparagine (Q/N)-rich prions and the neurotoxicity of polyQ-expanded proteins involve complex aggregation-prone structural transitions, commonly presumed to be forming β sheets. By analyzing sequences of interaction partners of these proteins, we discovered a recurrent presence of coiled-coil domains both in the partners and in segments that flank or overlap Q/N-rich and polyQ domains. Since coiled coils can mediate protein interactions and multimerization, we studied their possible involvement in Q/N-rich and polyQ aggregations. Using circular dichroism and chemical crosslinking, we found that Q/N-rich and polyQ peptides form α-helical coiled coils in vitro and assemble into multimers. Using structure-guided mutagenesis, we found that coiled-coil domains modulate in vivo properties of two Q/N-rich prions and polyQ-expanded huntingtin. Mutations that disrupt coiled coils impair aggregation and activity, whereas mutations that enhance coiled-coil propensity promote aggregation. These findings support a coiled-coil model for the functional switch of Q/N-rich prions and for the pathogenesis of polyQ-expansion diseases

A Combined Cell-Worm Approach to Search for Compounds Counteracting the Toxicity of Tau Oligomers In Vivo

A clear relationship between the tau assemblies and toxicity has still to be established. To correlate the tau conformation with its proteotoxic effect in vivo, we developed an innovative cell-worm-based approach. HEK293 cells expressing tau P301L under a tetracycline-inducible system (HEK T-Rex) were employed to produce different tau assemblies whose proteotoxic potential was evaluated using C. elegans. Lysates from cells induced for five days significantly reduced the worm’s locomotor activity. This toxic effect was not related to the total amount of tau produced by cells or to its phosphorylation state but was related to the formation of multimeric tau assemblies, particularly tetrameric ones. We investigated the applicability of this approach for testing compounds acting against oligomeric tau toxicity, using doxycycline (Doxy) as a prototype drug. Doxy affected tau solubility and promoted the disassembly of already formed toxic aggregates in lysates of cells induced for five days. These effects translated into a dose-dependent protective action in C. elegans. These findings confirm the validity of the combined HEK T-Rex cells and the C. elegans-based approach as a platform for pharmacological screening