Establishment of Method for the Determination of Aggregated α-Synuclein in DLB Patient Using RT-QuIC Assay.

BACKGROUND: The accumulation of aggregated α-synuclein (αSyn) is known as one of the critical reasons to exhibit their variable molecular pathologies and phenotypes in synucleinopathies. Recent studies suggested that the real-time quaking-induced conversion (RT-QuIC) assay is one of the potential methods to detect these αSyn aggregates and could detect the aggregated αSyn in the brain tissue and cerebrospinal fluid (CSF) using the propensity of the prion-like oligomerization. OBJECTIVE: We tried to optimize the αSyn RT-QuIC assay based on the aggregation of αSyn in brain samples of synucleinopathies by comparing the conditions of the recently reported αSyn RTQuIC assays. METHODS: This study applied a highly sensitive RT-QuIC assay using recombinant αSyn (rαSyn) to detect aggregated αSyn in the brain tissue from dementia with Lewy bodies (DLB). RESULTS: This study compared αSyn RT-QuIC assays under conditions such as beads, rαSyn as a substrate, reaction buffers, and fluorescence detectors. We observed that the addition of beads and the use of 6x His-tagged rαSyn as a substrate help to obtain higher positive responses from αSyn RT-QuIC assay seeding with brain homogenate (BH) of DLB and phosphate buffer-based reaction showed higher positive responses than HEPES buffer-based reaction on both fluorescent microplate readers. We also observed that the DLB BHs gave positive responses within 15-25h, which is faster high positive responses than recently reported assays. CONCLUSION: This established αSyn RT-QuIC assay will be able to apply to the early clinical diagnosis of αSyn aggregates-related diseases in various biofluids such as CSF.

The frequency of Merkel cell polyomavirus in whole blood from immunocompetent and immunosuppressed patients with kidney disease and healthy donors.

Merkel cell polyomavirus (MCPyV) is a rare, aggressive and related to human diseases in immunocompromised patients. MCPyV has been detected in skin neoplasms, various cancers, immunosuppressed patients and immunocompetent individuals. Several studies have confirmed the presence of MCPyV in patients with kidney dysfunction, such as kidney transplant (KTx) and long-term dialysis patients. The aims of this study were to quantify and compare the frequency of MCPyV in whole blood samples from immunocompetent and immunosuppressed patients and healthy blood donors and to compare MCPyV genotypes in a Korean population. DNA from Groups 1, 2, and 3 was screened for MCPyV using polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) with primer pairs targeting two regions of the large T-antigen. Thirteen of 122 whole-blood samples (12.7%) were positive for MCPyV. The virus was detected in the three groups of patients and healthy donors; specifically, in 5 of 30 (16.7%) KTx patients (Group 1), 6 of 52 (11.5%) dialysis patients (Group 2), and 4 of 40 (10%) healthy donors (Group 3). Low viral DNA loads 4.4-18 copies/µl were observed using qPCR DNA sequences from the two MCPyV-LT regions, which showed high homology with MCPyV sequences belonging to the TKS strain from Japan rather than the Chinese/European/North American strains. The MCPyV DNA was similarly amplified in whole blood from immunocompetent and immunosuppressed patients and healthy donors. This virus may be involved in establishing the persistence of infected peripheral leukocytes in the host, based on the incidence of detection of MCPyV DNA in blood samples from immunocompromised and immunocompetent subjects. This study is the first to identify a Korean MCPyV strain in whole-blood samples from Korean patients with kidney disease and healthy individuals.

Phosphatidylinositol-glycan-phospholipase D is involved in neurodegeneration in prion disease.

PrPSc is formed from a normal glycosylphosphatidylinositol (GPI)-anchored prion protein (PrPC) by a posttranslational modification. Most GPI-anchored proteins have been shown to be cleaved by GPI phospholipases. Recently, GPI-phospholipase D (GPI-PLD) was shown to be a strictly specific enzyme for GPI anchors. To investigate the involvement of GPI-PLD in the processes of neurodegeneration in prion diseases, we examined the mRNA and protein expression levels of GPI-PLD in the brains of a prion animal model (scrapie), and in both the brains and cerebrospinal fluids (CSF) of sporadic and familial Creutzfeldt-Jakob disease (CJD) patients. We found that compared with controls, the expression of GPI-PLD was dramatically down-regulated in the brains of scrapie-infected mice, especially in the caveolin-enriched membrane fractions. Interestingly, the observed decrease in GPI-PLD expression levels began at the same time that PrPSc began to accumulate in the infected brains and this decrease was also observed in both the brain and CSF of CJD patients; however, no differences in expression were observed in either the brains or CSF specimens from Alzheimer's disease patients. Taken together, these results suggest that the down-regulation of GPI-PLD protein may be involved in prion propagation in the brains of prion diseases.

Lack of association between 14-3-3 beta gene (YWHAB) polymorphisms and sporadic Creutzfeldt-Jakob disease (CJD).

14-3-3 proteins are highly abundant in brain tissue. The presence of 14-3-3 at elevated levels in the cerebrospinal fluid has been considered as a biomarker for sporadic Creutzfeldt-Jakob disease (CJD). Recent studies showed that 14-3-3 beta protein interacts with the N-terminal amino acids 1-38 and with the central hydrophobic amino acids 106-126 of prion protein. This interaction may indicate a role of 14-3-3 beta in the biological function of PrP and in the pathogenesis of prion disease. An association between the polymorphisms of 14-3-3 beta gene (YWHAB) and prion disease has not been reported previously. In order to investigate whether YWHAB polymorphisms are associated with sporadic CJD in the Korean population, we compared genotype distribution and allele frequencies of six YWHAB polymorphisms in 244 sporadic CJD patients and 219 healthy Koreans. Of six polymorphisms identified, four single nucleotide polymorphisms (SNPs) were known previously (c.60A>C, c.685-120G>A, c.685-89G>A, 92G>A) and two SNPs were novel (c.185T>A and c.377A>C). Two novel polymorphisms were identified within 3'-untranslated region of exon 6. We could not find significant differences in genotype and allele frequencies of the six YWHAB polymorphisms between the controls and sporadic CJD patients. These results indicate that these six YWHAB polymorphisms are not associated with the genetic susceptibility to sporadic CJD. This is the first genetic association study of YWHAB in sporadic CJD.

The first report of RPSA polymorphisms, also called 37/67 kDa LRP/LR gene, in sporadic Creutzfeldt-Jakob disease (CJD).

BACKGROUND: Although polymorphisms of PRNP, the gene encoding prion protein, are known as a determinant affecting prion disease susceptibility, other genes also influence prion incubation time. This finding offers the opportunity to identify other genetic or environmental factor (s) modulating susceptibility to prion disease. Ribosomal protein SA (RPSA), also called 37 kDa laminin receptor precursor (LRP)/67 kDa laminin receptor (LR), acts as a receptor for laminin, viruses and prion proteins. The binding/internalization of prion protein is dependent for LRP/LR. METHODS: To identify other susceptibility genes involved in prion disease, we performed genetic analysis of RPSA. For this case-control study, we included 180 sporadic Creutzfeldt-Jakob disease (CJD) patients and 189 healthy Koreans. We investigated genotype and allele frequencies of polymorphism on RPSA by direct sequencing or restriction fragment length polymorphism (RFLP) analysis. RESULTS: We observed four single nucleotide polymorphisms (SNPs), including -8T>C (rs1803893) in the 5'-untranslated region (UTR) of exon 2, 134-32C>T (rs3772138) in the intron, 519G>A (rs2269350) in the intron and 793+58C>T (rs2723) in the intron on the RPSA. The 519G>A (at codon 173) is located in the direct PrP binding site. The genotypes and allele frequencies of the RPSA polymorphisms showed no significant differences between the controls and sporadic CJD patients. CONCLUSION: These results suggest that these RPSA polymorphisms have no direct influence on the susceptibility to sporadic CJD. This was the first genetic association study of the polymorphisms of RPSA gene with sporadic CJD.