Scrapie Affects the Maturation Cycle and Immune Complex Trapping by Follicular Dendritic Cells in Mice

Transmissible spongiform encephalopathies (TSEs) or prion diseases are infectious neurological disorders of man and animals, characterised by abnormal disease-associated prion protein (PrPd) accumulations in the brain and lymphoreticular system (LRS). Prior to neuroinvasion, TSE agents often accumulate to high levels within the LRS, apparently without affecting immune function. However, our analysis of scrapie-affected sheep shows that PrPd accumulations within the LRS are associated with morphological changes to follicular dendritic cells (FDCs) and tingible body macrophages (TBMs). Here we examined FDCs and TBMs in the mesenteric lymph nodes (MLNs) of scrapie-affected mice by light and electron microscopy. In MLNs from uninfected mice, FDCs could be morphologically categorised into immature, mature and regressing forms. However, in scrapie-affected MLNs this maturation cycle was adversely affected. FDCs characteristically trap and retain immune complexes on their surfaces, which they display to B-lymphocytes. In scrapie-affected MLNs, some FDCs were found where areas of normal and abnormal immune complex retention occurred side by side. The latter co-localised with PrPd plasmalemmal accumulations. Our data suggest this previously unrecognised morphology represents the initial stage of an abnormal FDC maturation cycle. Alterations to the FDCs included PrPd accumulation, abnormal cell membrane ubiquitin and excess immunoglobulin accumulation. Regressing FDCs, in contrast, appeared to lose their membrane-attached PrPd. Together, these data suggest that TSE infection adversely affects the maturation and regression cycle of FDCs, and that PrPd accumulation is causally linked to the abnormal pathology observed. We therefore support the hypothesis that TSEs cause an abnormality in immune function

A Camelid Anti-PrP Antibody Abrogates PrPSc Replication in Prion-Permissive Neuroblastoma Cell Lines

The development of antibodies effective in crossing the blood brain barrier (BBB), capable of accessing the cytosol of affected cells and with higher affinity for PrPSc would be of paramount importance in arresting disease progression in its late stage and treating individuals with prion diseases. Antibody-based therapy appears to be the most promising approach following the exciting report from White and colleagues, establishing the “proof-of-principle” for prion-immunotherapy. After passive transfer, anti-prion antibodies were shown to be very effective in curing peripheral but not central rodent prion disease, due to the fact that these anti-prion antibodies are relatively large molecules and cannot therefore cross the BBB. Here, we show that an anti-prion antibody derived from camel immunised with murine scrapie material adsorbed to immunomagnetic beads is able to prevent infection of susceptible N2a cells and cure chronically scrapie-infected neuroblastoma cultures. This antibody was also shown to transmigrate across the BBB and cross the plasma membrane of neurons to target cytosolic PrPC. In contrast, treatment with a conventional anti-prion antibody derived from mouse immunised with recombinant PrP protein was unable to prevent recurrence of PrPSc replication. Furthermore, our camelid antibody did not display any neurotoxic effects following treatment of susceptible N2a cells as evidenced by TUNEL staining. These findings demonstrate the potential use of anti-prion camelid antibodies for the treatment of prion and other related diseases via non-invasive means

Binding-Folding Induced Regulation of AF1 Transactivation Domain of the Glucocorticoid Receptor by a Cofactor That Binds to Its DNA Binding Domain

Intrinsically disordered (ID) regions of proteins commonly exist within transcription factors, including the N-terminal domain (NTD) of steroid hormone receptors (SHRs) that possesses a powerful activation function, AF1 region. The mechanisms by which SHRs pass signals from a steroid hormone to control gene expression remain a central unresolved problem. The role of N-terminal activation function AF1, which exists in an intrinsically disordered (ID) conformation, in this process is of immense importance. It is hypothesized that under physiological conditions, ID AF1 undergoes disorder/order transition via inter- and intra-molecular communications, which allows AF1 surfaces to interact with specific co-regulatory proteins, critical for the final outcome of target gene expression regulated by SHRs. However, the means by which AF1 acquires functionally folded conformations is not well understood. In this study, we tested whether binding of jun dimerization protein 2 (JDP2) within the DNA binding domain (DBD) of the glucocorticoid receptor (GR) leads to acquisition of functionally active structure in its AF1/NTD. Our results show that signals mediated from GR DBD:JDP2 interactions in a two domain GR fragment, consisting of the entire NTD and little beyond DBD, significantly increased secondary/tertiary structure formation in the NTD/AF1. This increased structure formation facilitated AF1’s interaction with specific co-regulatory proteins and subsequent glucocorticoid response element-mediated AF1 promoter:reporter activity. These results support the hypothesis that inter- and intra-molecular signals give a functionally active structure(s) to the GR AF1, which is important for its transcriptional activity

Pathogenic alpha-synuclein aggregates preferentially bind to mitochondria and affect cellular respiration

Abstract Misfolded alpha-synuclein (αSyn) is a major constituent of Lewy bodies and Lewy neurites, which are pathological hallmarks of Parkinson’s disease (PD). The contribution of αSyn to PD is well established, but the detailed mechanism remains obscure. Using a model in which αSyn aggregation in primary neurons was seeded by exogenously added, preformed αSyn amyloid fibrils (PFF), we found that a majority of pathogenic αSyn (indicated by serine 129 phosphorylated αSyn, ps-αSyn) was membrane-bound and associated with mitochondria. In contrast, only a minuscule amount of physiological αSyn was mitochondrial bound. In vitro, αSyn PFF displayed a stronger binding to purified mitochondria than did αSyn monomer, revealing a preferential mitochondria binding by aggregated αSyn. This selective mitochondrial ps-αSyn accumulation was confirmed in other neuronal and animal αSyn aggregation models that do not require exogenously added PFF and, more importantly, in postmortem brain tissues of patients suffering from PD and other neurodegenerative diseases with αSyn aggregation (α-synucleinopathies). We also showed that the mitochondrial ps-αSyn accumulation was accompanied by defects in cellular respiration in primary neurons, suggesting a link to mitochondrial dysfunction. Together, our results show that, contrary to physiological αSyn, pathogenic αSyn aggregates preferentially bind to mitochondria, indicating mitochondrial dysfunction as the common downstream mechanism for α-synucleinopathies. Our findings suggest a plausible model explaining the formation and the peculiar morphology of Lewy body and reveal that disrupting the interaction between ps-αSyn and the mitochondria is a therapeutic target for α-synucleinopathies.https://deepblue.lib.umich.edu/bitstream/2027.42/148288/1/40478_2019_Article_696.pd

Detection of Sub-Clinical CWD Infection in Conventional Test-Negative Deer Long after Oral Exposure to Urine and Feces from CWD+ Deer

Chronic wasting disease (CWD) of cervids is a prion disease distinguished by high levels of transmissibility, wherein bodily fluids and excretions are thought to play an important role. Using cervid bioassay and established CWD detection methods, we have previously identified infectious prions in saliva and blood but not urine or feces of CWD+ donors. More recently, we identified very low concentrations of CWD prions in urine of deer by cervid PrP transgenic (Tg[CerPrP]) mouse bioassay and serial protein misfolding cyclic amplification (sPMCA). This finding led us to examine further our initial cervid bioassay experiments using sPMCA. distribution in these animals.Various neural and lymphoid tissues from conventional test-negative deer were reanalyzed for CWD prions by sPMCA and cervid transgenic mouse bioassay in parallel with appropriate tissue-matched positive and negative controls. was amplified from both lymphoid and neural tissues of positive control deer but not from identical tissues of negative control deer.Detection of subclinical infection in deer orally exposed to urine and feces (1) suggests that a prolonged subclinical state can exist, necessitating observation periods in excess of two years to detect CWD infection, and (2) illustrates the sensitive and specific application of sPMCA in the diagnosis of low-level prion infection. Based on these results, it is possible that low doses of prions, e.g. following oral exposure to urine and saliva of CWD-infected deer, bypass significant amplification in the LRS, perhaps utilizing a neural conduit between the alimentary tract and CNS, as has been demonstrated in some other prion diseases

Isolation of Phosphatidylethanolamine as a Solitary Cofactor for Prion Formation in the Absence of Nucleic Acids

Infectious prions containing the pathogenic conformer of the mammalian prion protein (PrP(Sc)) can be produced de novo from a mixture of the normal conformer (PrP(C)) with RNA and lipid molecules. Recent reconstitution studies indicate that nucleic acids are not required for the propagation of mouse prions in vitro, suggesting the existence of an alternative prion propagation cofactor in brain tissue. However, the identity and functional properties of this unique cofactor are unknown. Here, we show by purification and reconstitution that the molecule responsible for the nuclease-resistant cofactor activity in brain is endogenous phosphatidylethanolamine (PE). Synthetic PE alone facilitates conversion of purified recombinant (rec)PrP substrate into infectious recPrP(Sc) molecules. Other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol, were unable to facilitate recPrP(Sc) formation in the absence of RNA. PE facilitated the propagation of PrP(Sc) molecules derived from all four different animal species tested including mouse, suggesting that unlike RNA, PE is a promiscuous cofactor for PrP(Sc) formation in vitro. Phospholipase treatment abolished the ability of brain homogenate to reconstitute the propagation of both mouse and hamster PrP(Sc) molecules. Our results identify a single endogenous cofactor able to facilitate the formation of prions from multiple species in the absence of nucleic acids or other polyanions

Co-Infection with the Friend Retrovirus and Mouse Scrapie Does Not Alter Prion Disease Pathogenesis in Susceptible Mice

Prion diseases are fatal, transmissible neurodegenerative diseases of the central nervous system. An abnormally protease-resistant and insoluble form (PrPSc) of the normally soluble protease-sensitive host prion protein (PrPC) is the major component of the infectious prion. During the course of prion disease, PrPSc accumulates primarily in the lymphoreticular and central nervous systems. Recent studies have shown that co-infection of prion-infected fibroblast cells with the Moloney murine leukemia virus (Mo-MuLV) strongly enhanced the release and spread of scrapie infectivity in cell culture, suggesting that retroviral coinfection might significantly influence prion spread and disease incubation times in vivo. We now show that another retrovirus, the murine leukemia virus Friend (F-MuLV), also enhanced the release and spread of scrapie infectivity in cell culture. However, peripheral co-infection of mice with both Friend virus and the mouse scrapie strain 22L did not alter scrapie disease incubation times, the levels of PrPSc in the brain or spleen, or the distribution of pathological lesions in the brain. Thus, retroviral co-infection does not necessarily alter prion disease pathogenesis in vivo, most likely because of different cell-specific sites of replication for scrapie and F-MuLV

Cofactor Molecules Maintain Infectious Conformation and Restrict Strain Properties in Purified Prions

Prions containing misfolded prion protein (PrP(Sc)) can be formed with cofactor molecules using the technique of serial protein misfolding cyclic amplification. However, it remains unknown whether cofactors materially participate in maintaining prion conformation and infectious properties. Here we show that withdrawal of cofactor molecules during serial propagation of purified recombinant prions caused adaptation of PrP(Sc) structure accompanied by a reduction in specific infectivity of >10(5)-fold, to undetectable levels, despite the ability of adapted “protein-only” PrP(Sc) molecules to self-propagate in vitro. We also report that changing only the cofactor component of a minimal reaction substrate mixture during serial propagation induced major changes in the strain properties of an infectious recombinant prion. Moreover, propagation with only one functional cofactor (phosphatidylethanolamine) induced the conversion of three distinct strains into a single strain with unique infectious properties and PrP(Sc) structure. Taken together, these results indicate that cofactor molecules can regulate the defining features of mammalian prions: PrP(Sc) conformation, infectivity, and strain properties. These findings suggest that cofactor molecules likely are integral components of infectious prions

Inhibiting the mitochondrial pyruvate carrier does not ameliorate synucleinopathy in the absence of inflammation or metabolic deficits

Epidemiological studies suggest a link between type-2 diabetes and Parkinson’s disease (PD) risk. Treatment of type-2 diabetes with insulin sensitizing drugs lowers the risk of PD. We previously showed that the insulin sensitizing drug, MSDC-0160, ameliorates pathogenesis in some animal models of PD. MSDC-0160 reversibly binds the mitochondrial pyruvate carrier (MPC) protein complex, which has an anti-inflammatory effect and restores metabolic deficits. Since PD is characterized by the deposition of α-synuclein (αSyn), we hypothesized that inhibiting the MPC might directly inhibit αSyn aggregation in vivo in mammals. To answer if modulation of MPC can reduce the development of αSyn assemblies, and reduce neurodegeneration, we treated two chronic and progressive mouse models; a viral vector-based αSyn overexpressing model and a pre-formed fibril (PFF) αSyn seeding model with MSDC-0160. These two models present distinct types of αSyn pathology but lack inflammatory or autophagy deficits. Contrary to our hypothesis, we found that a modulation of MPC in these models did not reduce the accumulation of αSyn aggregates or mitigate neurotoxicity. Instead, MSDC-0160 changed the post-translational modification and aggregation features of αSyn. These results are consistent with the lack of a direct effect of MPC modulation on synuclein clearance in these models

Loss of One Engrailed1 Allele Enhances Induced α-Synucleinopathy

Parkinson’s disease (PD) is a synucleinopathy that has multiple neuropathological characteristics, with nigrostriatal dopamine system degeneration being a core feature. Current models of PD pathology typically fail to recapitulate several attributes of the pathogenic process and neuropathology. We aimed to define the effects of combining a mouse model exhibiting multiple PD-like changes with intrastriatal injections of α-synuclein (α-syn) pre-formed fibril (PFFs) aggregates. We employed the heterozygous Engrailed 1 (En1+/–) mouse that features several pathophysiological hallmarks of clinical PD.La enfermedad de Parkinson (EP) es una sinucleinopatía que tiene múltiples características neuropatológicas, siendo la degeneración del sistema dopaminérgico nigroestriatal una característica central. Los modelos actuales de patología de la EP generalmente no logran recapitular varios atributos del proceso patogénico y la neuropatología. Nuestro objetivo fue definir los efectos de combinar un modelo de ratón que presentaba múltiples cambios similares a los de la EP con inyecciones intraestriatales de agregados de fibrillas preformadas (PFF) de α-sinucleína (α-syn). Empleamos el ratón heterocigoto Engrailed 1 (En1+/–) que presenta varias características fisiopatológicas de la EP clínica