ZyFISH: A Simple, Rapid and Reliable Zygosity Assay for Transgenic Mice

Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations

Novel mutation of the PRNP gene of a clinical CJD case

BACKGROUND: Transmissible spongiform encephalopathies (TSEs), a group of neurodegenerative diseases, are thought to be caused by an abnormal isoform of a naturally occurring protein known as cellular prion protein, PrP(C). The abnormal form of prion protein, PrP(Sc )accumulates in the brain of affected individuals. Both isoforms are encoded by the same prion protein gene (PRNP), and the structural changes occur post-translationally. Certain mutations in the PRNP gene result in genetic TSEs or increased susceptibility to TSEs. CASE PRESENTATION: A 70 year old woman was admitted to the hospital with severe confusion and inability to walk. Relatives recognized memory loss, gait and behavioral disturbances over a six month period prior to hospitalization. Neurological examination revealed Creutzfeldt-Jakob disease (CJD) related symptoms such as incontinence, Babinski sign and myoclonus. EEG showed periodic sharp waves typical of sporadic CJD and cerebrospinal fluid analysis (CSF) was positive for the presence of the 14-3-3-protein. As the disease progressed the patient developed akinetic mutism and died in the tenth month after onset of the disease symptoms. Unfortunately, no autopsy material was available. PRNP sequencing showed the occurrence of a point mutation on one allele at codon 193, which is altered from ACC, coding for a threonine, to ATC, encoding an isoleucine (T193I). CONCLUSION: Here we report a novel mutation of the PRNP gene found in an elderly female patient resulting in heterozygosity for isoleucine and threonine at codon 193, in which normally homozygosity for threonine is expected (T193). The patient presented typical clinical symptoms of CJD. EEG findings and the presence of the 14-3-3 protein in the CSF, contributed to CJD diagnosis, allowing the classification of this case as a probable CJD according to the World Health Organization (WHO) accepted criteria

Insights into Spatial Sensitivities of Ice Mass Response to Environmental Change from the SeaRISE Ice Sheet Modeling Project I: Antarctica

Atmospheric, oceanic, and subglacial forcing scenarios from the Sea-level Response to Ice Sheet Evolution (SeaRISE) project are applied to six three-dimensional thermomechanical ice-sheet models to assess Antarctic ice sheet sensitivity over a 500 year timescale and to inform future modeling and field studies. Results indicate (i) growth with warming, except within low-latitude basins (where inland thickening is outpaced by marginal thinning); (ii) mass loss with enhanced sliding (with basins dominated by high driving stresses affected more than basins with low-surface-slope streaming ice); and (iii) mass loss with enhanced ice shelf melting (with changes in West Antarctica dominating the signal due to its marine setting and extensive ice shelves; cf. minimal impact in the Terre Adelie, George V, Oates, and Victoria Land region of East Antarctica). Ice loss due to dynamic changes associated with enhanced sliding and/or sub-shelf melting exceeds the gain due to increased precipitation. Furthermore, differences in results between and within basins as well as the controlling impact of sub-shelf melting on ice dynamics highlight the need for improved understanding of basal conditions, grounding-zone processes, ocean-ice interactions, and the numerical representation of all three

The 5′ Flanking Region and Intron1 of the Bovine Prion Protein Gene (PRNP) Are Responsible for Negative Feedback Regulation of the Prion Protein

Transcription factors regulate gene expression by controlling the transcription rate. Some genes can repress their own expression to prevent over production of the corresponding protein, although the mechanism and significance of this negative feedback regulation remains unclear. In the present study, we describe negative feedback regulation of the bovine prion protein (PrP) gene PRNP in Japanese Black cattle. The PrP-expressing plasmid pEF-boPrP and luciferase-expressing plasmids containing the partial promoter fragment of PRNP incorporating naturally occurring single-nucleotide or insertion/deletion polymorphisms were transfected into N2a cells. Transfection of pEF-boPrP induced PrP overexpression and decreased the promoter activity of PRNP in the wild-type haplotype (23-bp Del, 12-bp Del, and −47C). Reporter gene assays further demonstrated that the 12- and 23-bp Ins/Del polymorphisms, which are thought to be associated with Sp1 (Specific protein 1) and RP58 (Repressor Protein with a predicted molecular mass of 58 kDa), in intron1 and the upstream region, respectively, and an additional polymorphism (−47C→A) in the Sp1-binding site responded differently to PrP overexpression. With the −47C SNP, the presence of the Del in either the 23-bp Ins/Del or the 12-bp Ins/Del allele was essential for the negative feedback caused by PrP overexpression. Furthermore, deletion mutants derived from the wild-type haplotype showed that nucleotides −315 to +2526, which include the 5′-flanking region and exon1, were essential for the response. These results indicate that certain negative feedback response elements are located in these sequences, suggesting that regulation by transcription factors such as Sp1 and RP58 may contribute to the negative feedback mechanism of PRNP

Idebenone and Resveratrol Extend Lifespan and Improve Motor Function of HtrA2 Knockout Mice

Heterozygous loss-of-function mutation of the human gene for the mitochondrial protease HtrA2 has been associated with increased risk to develop mitochondrial dysfunction, a process known to contribute to neurodegenerative disorders such as Huntington's disease (HD) and Parkinson's disease (PD). Knockout of HtrA2 in mice also leads to mitochondrial dysfunction and to phenotypes that resemble those found in neurodegenerative disorders and, ultimately, lead to death of animals around postnatal day 30. Here, we show that Idebenone, a synthetic antioxidant of the coenzyme Q family, and Resveratrol, a bioactive compound extracted from grapes, are both able to ameliorate this phenotype. Feeding HtrA2 knockout mice with either compound extends lifespan and delays worsening of the motor phenotype. Experiments conducted in cell culture and on brain tissue of mice revealed that each compound has a different mechanism of action. While Idebenone acts by downregulating the integrated stress response, Resveratrol acts by attenuating apoptosis at the level of Bax. These activities can account for the delay in neuronal degeneration in the striata of these mice and illustrate the potential of these compounds as effective therapeutic approaches against neurodegenerative disorders such as HD or PD

PrP(Sc)-specific antibodies with the ability to immunodetect prion oligomers.

The development of antibodies with binding capacity towards soluble oligomeric forms of PrPSc recognised in the aggregation process in early stage of the disease would be of paramount importance in diagnosing prion diseases before extensive neuropathology has ensued. As blood transfusion appears to be efficient in the transmission of the infectious prion agent, there is an urgent need to develop reagents that would specifically recognize oligomeric forms of the abnormally folded prion protein, PrPSc.To that end, we show that anti-PrP monoclonal antibodies (called PRIOC mAbs) derived from mice immunised with native PrP-coated microbeads are able to immunodetect oligomers/multimers of PrPSc. Oligomer-specific immunoreactivity displayed by these PRIOC mAbs was demonstrated as large aggregates of immunoreactive deposits in prion-permissive neuroblastoma cell lines but not in equivalent non-infected or prn-p(0/0) cell lines. In contrast, an anti-monomer PrP antibody displayed diffuse immunoreactivity restricted to the cell membrane. Furthermore, our PRIOC mAbs did not display any binding with monomeric recombinant and cellular prion proteins but strongly detected PrPSc oligomers as shown by a newly developed sensitive and specific ELISA. Finally, PrioC antibodies were also able to bind soluble oligomers formed of Aβ and α-synuclein. These findings demonstrate the potential use of anti-prion antibodies that bind PrPSc oligomers, recognised in early stage of the disease, for the diagnosis of prion diseases in blood and other body fluids

Frequencies of polymorphisms associated with BSE resistance differ significantly between Bos taurus, Bos indicus, and composite cattle

<p>Abstract</p> <p>Background</p> <p>Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases that affect several mammalian species. At least three factors related to the host prion protein are known to modulate susceptibility or resistance to a TSE: amino acid sequence, atypical number of octapeptide repeats, and expression level. These factors have been extensively studied in breeds of <it>Bos taurus </it>cattle in relation to classical bovine spongiform encephalopathy (BSE). However, little is currently known about these factors in <it>Bos indicus </it>purebred or <it>B. indicus </it>× <it>B. taurus </it>composite cattle. The goal of our study was to establish the frequency of markers associated with enhanced susceptibility or resistance to classical BSE in <it>B. indicus </it>purebred and composite cattle.</p> <p>Results</p> <p>No novel or TSE-associated <it>PRNP</it>-encoded amino acid polymorphisms were observed for <it>B. indicus </it>purebred and composite cattle, and all had the typical number of octapeptide repeats. However, differences were observed in the frequencies of the 23-bp and 12-bp insertion/deletion (indel) polymorphisms associated with two bovine <it>PRNP </it>transcription regulatory sites. Compared to <it>B. taurus</it>, <it>B. indicus </it>purebred and composite cattle had a significantly lower frequency of 23-bp insertion alleles and homozygous genotypes. Conversely, <it>B. indicus </it>purebred cattle had a significantly higher frequency of 12-bp insertion alleles and homozygous genotypes in relation to both <it>B. taurus </it>and composite cattle. The origin of these disparities can be attributed to a significantly different haplotype structure within each species.</p> <p>Conclusion</p> <p>The frequencies of the 23-bp and 12-bp indels were significantly different between <it>B. indicus </it>and <it>B. taurus </it>cattle. No other known or potential risk factors were detected for the <it>B. indicus </it>purebred and composite cattle. To date, no consensus exists regarding which bovine <it>PRNP </it>indel region is more influential with respect to classical BSE. Should one particular indel region and associated genotypes prove more influential with respect to the incidence of classical BSE, differences regarding overall susceptibility and resistance for <it>B. indicus </it>and <it>B. taurus </it>cattle may be elucidated.</p

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

Preconditioning Involves Selective Mitophagy Mediated by Parkin and p62/SQSTM1

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia (sI) in vitro and IPC of hearts to investigate the role of Parkin in mediating cardioprotection ex vivo and in vivo. In HL-1 cells, sI induced Parkin translocation to mitochondria and mitochondrial elimination. IPC induced Parkin translocation to mitochondria in Langendorff-perfused rat hearts and in vivo in mice subjected to regional IPC. Mitochondrial depolarization with an uncoupling agent similarly induced Parkin translocation to mitochondria in cells and Langendorff-perfused rat hearts. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports indicating a role for p62/SQSTM1 in mitophagy, we found that depletion of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to sI. While wild type mice showed p62 translocation to mitochondria and an increase in ubiquitination, Parkin knockout mice exhibited attenuated IPC-induced p62 translocation to the mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection