Gino Miele

A novel erythroid-specific marker of transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies (TSE) are a group of invariably fatal neurodegenerative diseases and include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in deer and elk, and Kuru disease, Creutzfeldt–Jakob disease (CJD) and variant CJD in humans. The pathological effects of disease occur predominantly in the CNS (central nervous system), where common hallmarks include vacuolation, gliosis, accumulation of a protease-resistant, abnormally folded isoform of the prion protein (PrPSc) and neuronal cell death. Lack of understanding of the molecular mechanisms underlying disease pathogenesis, particularly in non-CNS tissues, means that there are currently no effective strategies for early diagnosis or therapeutic intervention of TSEs. Here we report the first identification of a molecular marker that is easily detectable in readily accessible tissues. We demonstrate that a dramatic decrease in expression of a transcript specific to erythroid lineage cells is a common feature of TSEs. Our findings indicate a previously unrecognized role for involvement of the erythroid lineage in the etiology of TSE pathogenesis and should provide a new focus for research into diagnostic and therapeutic strategies.

Embryonic activation and developmental expression of the murine prion protein gene.

While it is well established that cellular prion protein (PrP(C)) expression is required for the development of transmissible spongiform encephalopathies (TSEs), the physiological function of PrP(C) has yet to be determined. A number of studies have examined PrP expression in different tissues and in the later stages of embryonic development. However, the relative levels of expression of PrP RNA and protein in tissues outside the central nervous system (CNS) is not well documented and the exact point of transcriptional activation of PrP during embryogenesis is unknown. We have studied PrP mRNA expression in murine embryos and both mRNA and protein expression in a variety of adult tissues. PrP RNA was detected at different levels in all tissues tested while PrP(C) protein was detectable in all adult tissues tested with the exception of kidney and liver. RNA and protein levels were also assessed at four points during postnatal brain development and levels of both were seen to increase with development. We also established that, during embryogenesis, induction of PrP RNA expression occurs between E8.5 and E9, during the period of transition from anaerobic to aerobic metabolism. Preliminary experiments investigating the effects of superoxide radicals on PrP expression in cultured neuroblastoma and astrocyte cells support the suggestion that PrP(C) forms part of a cellular antioxidant defense mechanism.

Gene expression changes during murine postnatal brain development

BackgroundFor most vertebrate organs and tissues, the majority of development occurs during embryogenesis, and postnatal changes are primarily concerned with growth. The central nervous system is unusual in that a considerable amount of morphological development, cell differentiation and acquisition of function, takes place during postnatal development. As yet, the molecular mechanisms underlying these complex developmental processes are not well understood. In order to identify markers for these developmental processes, we have analyzed the expression profiles, during postnatal murine brain development, of approximately 25,000 transcripts. This analysis, performed at day 1, day 10, day 20 and day 42 of postnatal development, identified a large number of developmentally regulated genes which we have assigned into three broad expression categories.ResultsExpression levels at four timepoints during postnatal murine brain development were established for approximately 25,000 gene transcripts. Approximately 1% of the genes examined displayed a developmentally regulated pattern of expression and we provide all the necessary information required to easily obtain molecular markers for a subset of these developmentally regulated transcripts. Of this subset, 61 showed increasing expression during development, 61 showed decreasing expression during development, and 9 exhibited a peak of expression during this period.ConclusionsA small percentage of the genes expressed in the postnatal developing brain show changes in expression level during the newborn to adult phase of development. It is likely that these developmentally regulated transcripts represent molecular markers for the complex developmental process occurring in the postnatal brain.

A Rapid Protocol for the Authentication of Isolated Differential Display RT-PCR CDNAs

The most time-consuming and problematic step in the overall DDRT-PCR technique is the confirmation that the isolated cDNA clone represents a differentially expressed gene. We have previously suggested that the majority of apparent false positives generated by DDRT-PCR do in fact result from the PCR reamplification of cDNA species which co-migrate with the cDNA of interest, and we have outlined a procedure to effectively eliminate these from further study. However, in situations where RNA is limiting, it is still desirable to confirm that a purified cDNA amplicon does, in fact, represent the originally observed differentially expressed gene prior to embarking on expression studies.

Reply to “Is EDRF a specific marker for TSEs?”

Miele and Clinton reply—Although we chose to limit speculation on the mechanism underlying our recent findings, we feel that the suggestion by Maury represents one of a number of reasonable interpretations. Maury is correct in stating that variation in the levels of circulating cytokines might affect the numbers of erythroid progenitor cells in bone marrow, although current eviLETTERS TO THE EDITOR

Identification of disease markers by differential display: prion disease.

In order to identify molecular markers of prion disease in peripheral tissues, we used the differential display reverse-transcriptase polymerase chain reaction (DDRT-PCR) procedure to compare gene expression in spleens of infected and uninfected mice. In this study, we identified a novel erythroid-specific gene that was differentially expressed as a result of prion infection. We were able to demonstrate that a decrease in the expression levels of this transcript in hematopoietic tissues was a common feature of prion diseases. Our findings suggest a previously unknown role for the blood erythroid lineage in the development of prion diseases and should provide a new focus for research into diagnostic and therapeutic strategies.