Myelin Basic Protein Citrullination, a Hallmark of Central Nervous System Demyelination, Assessed by Novel Monoclonal Antibodies in Prion Diseases.

Myelin basic protein (MBP) citrullination by peptidylarginine deiminase (PAD) enzymes leads to incomplete protein-lipid bilayer interactions and vulnerability to proteolytic enzymes, resulting in disorganization of the myelin sheath in the central nervous system. Therefore, citrullinated MBP (citMBP) has been suggested as a hallmark of demyelination, but how citMBP is implicated in prion diseases remains unknown. For the first time, we developed mouse monoclonal anti-citMBP IgG1 (clones 1B8, 1H1, and 3C6) and IgM (clone 3G5) antibodies that recognize human citMBP at its R25, R122, and R130 residues and at its C-terminal region (or the corresponding sites in mouse MBP), respectively. Using a biochemical, immunohistochemical, and immunogold-silver staining for electron microscopy techniques, we found that MBP residue R23 (corresponding to human R25) was specifically citrullinated, was stained as intense punctae in the corpus callosum, the striatum, and the cerebellar white matter, and was predominantly localized in disorganized myelin in the brains of scrapie-infected mice. In the brains of Creutzfeldt-Jakob disease (CJD) patients, MBP residues R25, R122, and R130 were markedly citrullinated and were stained as fibrils and punctae. In particular, white matter regions, such as the midbrain and the medulla, exhibited high levels of citMBP compared to other regions. However, the high levels of citMBP were not correlated with PAD2 expression. The clone 3G5 recognized significantly increased expression of the 18.5 kDa and/or 21.5 kDa variants of MBP in prion disease. Our findings suggest that significantly increased levels of citMBP may reflect demyelinating neuropathology, and that these newly developed antibodies may be useful for identifying demyelination.

Cellular Prion Protein Combined with Galectin-3 and -6 Affects the Infectivity Titer of an Endogenous Retrovirus Assayed in Hippocampal Neuronal Cells.

Prion diseases are infectious and fatal neurodegenerative diseases which require the cellular prion protein, PrPC, for development of diseases. The current study shows that the PrPC augments infectivity and plaque formation of a mouse endogenous retrovirus, MuLV. We have established four neuronal cell lines expressing mouse PrPC, PrP+/+; two express wild type PrPC (MoPrPwild) and the other two express mutant PrPC (MoPrPmut). Infection of neuronal cells from various PrP+/+ and PrP-/- (MoPrPKO) lines with MuLV yielded at least three times as many plaques in PrP+/+ than in PrP-/-. Furthermore, among the four PrP+/+ lines, one mutant line, P101L, had at least 2.5 times as many plaques as the other three PrP+/+ lines. Plaques in P101L were four times larger than those in other PrP+/+ lines. Colocalization of PrP and CAgag was seen in MuLV-infected PrP+/+ cells. In the PrP-MuLV interaction, the involvement of galectin-3 and -6 was observed by immunoprecipitation with antibody to PrPC. These results suggest that PrPC combined with galectin-3 and -6 can act as a receptor for MuLV. P101L, the disease form of mutant PrPC results suggest the genetic mutant form of PrPC may be more susceptible to viral infection.

N(ε)-Carboxymethyl Modification of Lysine Residues in Pathogenic Prion Isoforms.

The most prominent hallmark of prion diseases is prion protein conversion and the subsequent deposition of the altered prions, PrP(Sc), at the pathological sites of affected individuals, particularly in the brain. A previous study has demonstrated that the N-terminus of the pathogenic prion isoform (PrP(Sc)) is modified with advanced glycation end products (AGEs), most likely at one or more of the three Lys residues (positions 23, 24, and 27) in the N-terminus (23KKRPKP28). The current study investigated whether N(ε)-(carboxymethyl)lysine (CML), a major AGE form specific to Lys residues produced by nonenzymatic glycation, is an AGE adduct of the N-terminus of PrP(Sc). We show that CML is linked to at least one Lys residue at the N-terminus of PrP(Sc) in 263K prion-infected hamster brains and at least one of the eight Lys residues (positions 101, 104, 106, 110, 185, 194, 204, and 220) in the proteinase K (PK)-resistant core region of PrP(Sc). The nonenzymatic glycation of the Lys residue(s) of PrP(Sc) with CML likely occurs in the widespread prion-deposit areas within infected brains, particularly in some of the numerous tyrosine hydroxylase-positive thalamic and hypothalamic nuclei. CML glycation does not occur in PrP(C) but is seen in the pathologic PrP(Sc) isoform. Furthermore, the modification of PrP(Sc) with CML may be closely involved in prion propagation and deposition in pathological brain areas.

Giant enhancement of the Raman response due to one-dimensional ZnO nanostructures.

We observed giant enhancement of the Raman intensity from 4-Mpy molecules adsorbed on semiconducting one-dimensional ZnO nanostructures, nanowires and nanocones, without involving any noble metals. Interestingly, the enhancement is strongly dependent on the geometry of ZnO nanostructures and can mainly be explained by the cavity-like structural resonance of the electric field. Our results can be applied to systematically create hot spots for Raman signal enhancement using one-dimensional semiconducting nanomaterials.

Tunicamycin induces paraptosis potentiated by inhibition of BRAFV600E in FRO anaplastic thyroid carcinoma cells.

BACKGROUND/AIM: The aim of the present study was to elucidate whether tunicamycin (TM) induces paraptosis as a cell death subroutine in anaplastic thyroid carcinoma (ATC) cells. MATERIALS AND METHODS: 8505C, CAL62 and FRO cells were used. After treatment of TM, cell survival and morphology were investigated. The effect of the BRAF(V600E) inhibitor PLX4032 in combination with TM was evaluated. RESULTS: In FRO cells, TM induced paraptosis characteristic of cytoplasmic vacuolation and endoplasmic reticulum (ER) swelling, which was not associated with caspase activation and ER stress. TM-induced paraptosis was ameliorated by pre-treatment with the translation inhibitor cycloheximide, while it was accelerated by pre-treatment with the proteasome inhibitor MG132. PLX4032 augmented TM-induced paraptosis. CONCLUSION: TM induces paraptosis relevant to de novo protein synthesis and proteasomal activity, and inhibition of BRAF(V600E) potentiates TM-induced paraptosis in FRO cells harboring BRAF(V600E).

Deficiency of prion protein induces impaired autophagic flux in neurons.

Normal cellular prion protein (PrP(C)) is highly expressed in the central nervous system. The Zürich I Prnp-deficient mouse strain did not show an abnormal phenotype in initial studies, however, in later studies, deficits in exploratory behavior and short- and long-term memory have been revealed. In the present study, numerous autophagic vacuoles were found in neurons from Zürich I Prnp-deficient mice. The autophagic accumulation in the soma of cortical neurons in Zürich I Prnp-deficient mice was observed as early as 3 months of age, and in the hippocampal neurons at 6 months of age. Specifically, there is accumulation of electron dense pigments associated with autophagy in the neurons of Zürich I Prnp-deficient mice. Furthermore, autophagic accumulations were observed as early as 3 months of age in the CA3 region of hippocampal and cerebral cortical neuropils. The autophagic vacuoles increased with age in the hippocampus of Zürich I Prnp-deficient mice at a faster rate and to a greater extent than in normal C57BL/6J mice, whereas the cortex exhibited high levels that were maintained from 3 months old in Zürich I Prnp-deficient mice. The pigmented autophagic accumulation is due to the incompletely digested material from autophagic vacuoles. Furthermore, a deficiency in PrP(C) may disrupt the autophagic flux by inhibiting autophagosome-lysosomal fusion. Overall, our results provide insight into the protective role of PrP(C) in neurons, which may play a role in normal behavior and other brain functions.

Stearoyl coenzyme A desaturase 1 is associated with hepatitis C virus replication complex and regulates viral replication.

UNLABELLED: The hepatitis C virus (HCV) life cycle is tightly regulated by lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have recently screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet formation using cell culture-grown HCV (HCVcc)-infected cells. We selected and characterized the gene encoding stearoyl coenzyme A (CoA) desaturase 1 (SCD1). siRNA-mediated knockdown or pharmacological inhibition of SCD1 abrogated HCV replication in both subgenomic replicon and Jc1-infected cells, while exogenous supplementation of either oleate or palmitoleate, products of SCD1 activity, resurrected HCV replication in SCD1 knockdown cells. SCD1 was coimmunoprecipitated with HCV nonstructural proteins and colocalized with both double-stranded RNA (dsRNA) and HCV nonstructural proteins, indicating that SCD1 is associated with HCV replication complex. Moreover, SCD1 was fractionated and enriched with HCV nonstructural proteins at detergent-resistant membrane. Electron microscopy data showed that SCD1 is required for NS4B-mediated intracellular membrane rearrangement. These data further support the idea that SCD1 is associated with HCV replication complex and that its products may contribute to the proper formation and maintenance of membranous web structures in HCV replication complex. Collectively, these data suggest that manipulation of SCD1 activity may represent a novel host-targeted antiviral strategy for the treatment of HCV infection. IMPORTANCE: Stearoyl coenzyme A (CoA) desaturase 1 (SCD1), a liver-specific enzyme, regulates hepatitis C virus (HCV) replication through its enzyme activity. HCV nonstructural proteins are associated with SCD1 at detergent-resistant membranes, and SCD1 is enriched on the lipid raft by HCV infection. Therein, SCD1 supports NS4B-mediated membrane rearrangement to provide a suitable microenvironment for HCV replication. We demonstrated that either genetic or chemical knockdown of SCD1 abrogated HCV replication in both replicon cells and HCV-infected cells. These findings provide novel mechanistic insights into the roles of SCD1 in HCV replication.

Dysfunction of mitochondrial dynamics in the brains of scrapie-infected mice.

Mitochondrial dysfunction is a common and prominent feature of many neurodegenerative diseases, including prion diseases; it is induced by oxidative stress in scrapie-infected animal models. In previous studies, we found swelling and dysfunction of mitochondria in the brains of scrapie-infected mice compared to brains of controls, but the mechanisms underlying mitochondrial dysfunction remain unclear. To examine whether the dysregulation of mitochondrial proteins is related to the mitochondrial dysfunction associated with prion disease, we investigated the expression patterns of mitochondrial fusion and fission proteins in the brains of ME7 prion-infected mice. Immunoblot analysis revealed that Mfn1 was up-regulated in both whole brain and specific brain regions, including the cerebral cortex and hippocampus, of ME7-infected mice compared to controls. Additionally, expression levels of Fis1 and Mfn2 were elevated in the hippocampus and the striatum, respectively, of the ME7-infected brain. In contrast, Dlp1 expression was significantly reduced in the hippocampus in the ME7-infected brain, particularly in the cytosolic fraction. Finally, we observed abnormal mitochondrial enlargement and histopathological change in the hippocampus of the ME7-infected brain. These observations suggest that the mitochondrial dysfunction, which is presumably caused by the dysregulation of mitochondrial fusion and fission proteins, may contribute to the neuropathological changes associated with prion disease.

Analysis of macular and peripapillary choroidal thickness in glaucoma patients by enhanced depth imaging optical coherence tomography.

BACKGROUND: To compare the macular and peripapillary choroidal thickness between normal and glaucoma eyes and find out factors related to choroidal thickness using enhanced depth imaging (EDI) of Heidelberg Spectralis SD-OCT. STUDY DESIGN: Cross-sectional transverse study. METHODS: A total of 108 glaucoma patients and 48 healthy controls were included in the analysis. Choroidal thickness was measured from 6 mm length radial B-scans at the macular and the optic nerve head by EDI OCT. Choroidal thickness was compared between normal controls, normal tension glaucoma (NTG) patients, and primary open-angle glaucoma (POAG) patients. Factors related to choroidal thickness were analyzed by regression analysis. RESULTS: There were no differences in average, temporal, nasal, superior, and inferior macular choroidal thickness between normal, NTG, and POAG eyes. The peripapillary thickness did not differ between normal and POAG eyes; however, average, temporal, nasal, superior, and inferior peripapillary choroidal thickness were significantly thinner in NTG eyes. Axial length (ß=-11.36, P<0.001) was the most significant factor associated with peripapillary choroidal thickness, followed by age (ß=-5.10, P<0.001). Glaucoma type (ß=-11.28, P<0.001) were also significantly associated with peripapillary choroidal thickness. CONCLUSIONS: Peripapillary choroidal thickness was significantly reduced in NTG eyes based on EDI OCT measurements in vivo.

Configuration-controlled Au nanocluster arrays on inverse micelle nano-patterns: versatile platforms for SERS and SPR sensors.

Nanopatterned 2-dimensional Au nanocluster arrays with controlled configuration are fabricated onto reconstructed nanoporous poly(styrene-block-vinylpyridine) inverse micelle monolayer films. Near-field coupling of localized surface plasmons is studied and compared for disordered and ordered core-centered Au NC arrays. Differences in evolution of the absorption band and field enhancement upon Au nanoparticle adsorption are shown. The experimental results are found to be in good agreement with theoretical studies based on the finite-difference time-domain method and rigorous coupled-wave analysis. The realized Au nanopatterns are exploited as substrates for surface-enhanced Raman scattering and integrated into Kretschmann-type SPR sensors, based on which unprecedented SPR-coupling-type sensors are demonstrated.

Contribution of macrophages to enhanced regenerative capacity of dorsal root ganglia sensory neurons by conditioning injury.

Although the central branches of the dorsal root ganglion (DRG) sensory neurons do not spontaneously regenerate, a conditioning peripheral injury can promote their regeneration. A potential role of macrophages in axonal regeneration was proposed, but it has not been critically addressed whether macrophages play an essential role in the conditioning injury model. After sciatic nerve injury (SNI) in rats, the number of macrophages in DRGs gradually increased by day 7. The increase persisted up to 28 d and was accompanied by upregulation of inflammatory mediators, including oncomodulin. A macrophage deactivator, minocycline, reduced the macrophage number and expressions of the inflammatory mediators. Molecular signatures of conditioning effects were abrogated by minocycline, and enhanced regenerative capacity was substantially attenuated both in vitro and in vivo. Delayed minocycline infusion abrogated the SNI-induced long-lasting heightened neurite outgrowth potential, indicating a role for macrophages in the maintenance of regenerative capacity. Intraganglionic cAMP injection also resulted in an increase in macrophages, and minocycline abolished the cAMP effect on neurite outgrowth. However, conditioned media (CM) from macrophages treated with cAMP did not exhibit neurite growth-promoting activity. In contrast, CM from neuron-macrophage cocultures treated with cAMP promoted neurite outgrowth greatly, highlighting a requirement for neuron-macrophage interactions for the induction of a proregenerative macrophage phenotype. The growth-promoting activity in the CM was profoundly attenuated by an oncomodulin neutralizing antibody. These results suggest that the neuron-macrophage interactions involved in eliciting a proregenerative phenotype in macrophages may be a novel target to induce long-lasting regenerative processes after axonal injuries in the CNS.

The Functional Role of Prion Protein (PrPC) on Autophagy.

Cellular prion protein (PrPC) plays an important role in the cellular defense against oxidative stress. However, the exact protective mechanism of PrPC is unclear. Autophagy is essential for survival, differentiation, development, and homeostasis in several organisms. Although the role that autophagy plays in neurodegenerative disease has yet to be established, it is clear that autophagy-induced cell death is observed in neurodegenerative disorders that exhibit protein aggregations. Moreover, autophagy can promote cell survival and cell death under various conditions. In this review, we describe the involvement of autophagy in prion disease and the effects of PrPC.

Subcellular localization of peptidylarginine deiminase 2 and citrullinated proteins in brains of scrapie-infected mice: nuclear localization of PAD2 and membrane fraction-enriched citrullinated proteins.

Peptidylarginine deiminase (PAD) and citrullinated proteins have emerged as key molecules in various human diseases, but detailed subcellular localizations of PAD2 and citrullinated proteins are poorly mapped in brain under normal and pathologic conditions. We performed subcellular fractionation and electron microscopic analysis using brains of normal and scrapie-infected mice. Peptidylarginine deiminase 2 was abundantly present in cytosol and weakly in microsomal and mitochondrial fractions and expression in these fractions was higher in brains of scrapie-infected mice. Despite relatively low PAD2 expression, in microsomal and mitochondrial fractions, citrullinated proteins were present at high levels in these fractions in scrapie-infected brains. Surprisingly, increased PAD2 expression and accumulated citrullinated proteins were also found in nuclear fractions in scrapie-infected brains. By electron microscopy, PAD2 and citrullinated proteins in scrapie-infected brains were widely distributed in most cellular compartments including mitochondria, endoplasmic reticulum, glial filaments, nuclei, and Golgi apparatus in astrocytes and hippocampal neurons. Taken together, we report for the first time the nuclear localization of PAD2 and the detailed subcellular localization of PAD2 and of citrullinated proteins in scrapie-infected brains. Our findings suggest that different subcellular compartmentalization of PAD2 and citrullinated proteins may have different physiological roles in normal and neurodegenerative conditions.

Association of endothelial nitric oxide synthase and mitochondrial dysfunction in the hippocampus of scrapie-infected mice.

The elevation of nitric oxide (NO) within the central nervous system (CNS) is known to be associated with the pathogenesis of neurodegenerative diseases such as HIV-associated dementia (HAD), brain ischemia, Parkinson's disease, and Alzheimer's disease. NO is enzymatically formed by the enzyme nitric oxide synthase (NOS). There are two forms of NOS, the constitutive and the inducible form. The constitutive form is present in endothelial cells (eNOS) and neurons (nNOS). The inducible form (iNOS) is expressed in various cell types including astroglia and microglia of the CNS. Using an animal model, we investigated the involvement of eNOS in the pathology of prion disease. We showed dramatic upregulation of eNOS immunoreactivity in reactive astroglial cells in the hippocampus in the prion disease animal model, scrapie in mice. Expression of eNOS was upregulated in cytosolic and mitochondrial fractions of whole brain. In the hippocampal region, eNOS was widely overexpressed in various components of the cell. We found that eNOS dramatically accumulated in hippocampal mitochondria and was particularly prevalent in structurally dysfunctional mitochondria. In association with the accumulation of eNOS in mitochondria, we showed that mitochondrial superoxide dismutase (Mn-SOD or SOD2), cytochrome c, and ATP activity were downregulated both in whole brain and in the hippocampal region. These results indicate that eNOS plays a role in the development of dysfunctional mitochondria and this, in turn, could induce some of the histopathological changes seen in prion diseases.

Physiological properties of astroglial cell lines derived from mice with high (SAMP8) and low (SAMR1, ICR) levels of endogenous retrovirus.

Previous studies have reported that various inbred SAM mouse strains differ markedly with regard to a variety of parameters, such as capacity for learning and memory, life spans and brain histopathology. A potential cause of differences seen in these strains may be based on the fact that some strains have a high concentration of infectious murine leukemia virus (MuLV) in the brain, whereas other strains have little or no virus. To elucidate the effect of a higher titer of endogenous retrovirus in astroglial cells of the brain, we established astroglial cell lines from SAMR1 and SAMP8 mice, which are, respectively, resistant and prone to deficit in learning and memory and shortened life span. MuLV-negative astroglial cell lines established from ICR mice served as controls. Comparison of these cell lines showed differences in: 1) levels of the capsid antigen CAgag in both cell lysates and culture media, 2) expression of genomic retroelements, 3) the number of virus particles, 4) titer of infectious virus, 5) morphology, 6) replication rate of cells in culture and final cell concentrations, 7) expression pattern of proinflammatory cytokine genes. The results show that the expression of MuLV is much higher in SAMP8 than SAMR1 astrocyte cultures and that there are physiological differences in astroglia from the 2 strains. These results raise the possibility that the distinct physiological differences between SAMP8 and SAMR1 are a function of activation of endogenous retrovirus.

The involvement of cellular prion protein in the autophagy pathway in neuronal cells.

Apoptosis and autophagy are main mechanisms of neuronal death involved in prion diseases. Serum deprivation can induce both pathways to cell death in various types of cells. To investigate whether PrP(C) is involved in autophagy pathway, we analyzed the level of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, by monitoring the conversion from LC3-I into LC3-II in Zürich I Prnp(-/-) hippocampal neuronal cells. We found that the expression level of LC3-II was increased in Prnp(-/-) compared to wild-type cells under serum deprivation. In electron microscopy, increased accumulation of autophagosomes in Prnp(-/-) cells was correlated with the increase in levels of LC3-II. Interestingly, this up-regulated autophagic activity was retarded by the introduction of PrP(C) into Prnp(-/-) cells but not by the introduction of PrP(C) lacking octapeptide repeat region. Thus, the octapeptide repeat region of PrP(C) may play a pivotal role in the control of autophagy exhibited by PrP(C) in neuronal cells.