Earlier onset of Alzheimer's disease: risk polymorphisms within PRNP, PRND, CYP46, and APOE genes.

We studied eight polymorphisms within APOE, PRNP, PRND, and CYP46 genes in 213 Polish late-onset patients with Alzheimer's disease (AD) and 171 non-demented elderly controls. A latent classification approach, grade-of-membership analysis, was taken to identify three extreme pure type risk sets defined by the probabilities of being affected with AD and for genotypes found at the examined genes. Sets I and II represented high intrinsic risk, having a higher density of various genotypes compared to set III, at low intrinsic risk. A gradient of onset age depending on membership in the risk sets was also observed. Logistic regression analysis showed that the highest risk for AD was found for individuals who co-inherited APOE epsilon4 allele, PRNP codon 129 homozygosity, PRND codon 174 Thr allele, and CYP46 rs754203 g allele. AD can be influenced by genetic profiles leading to appearance of the disease, composed of genes which separately evoke a little or unnoticeable effect. Moreover, there may be multiple sufficient risk sets for AD. Looking at multiple genes together rather than analyzing them individually, may improve identification of risk alleles.

Successful elimination of non-neural cells and unachievable elimination of glial cells by means of commonly used cell culture manipulations during differentiation of GFAP and SOX2 positive neural progenitors (NHA) to neuronal cells.

BACKGROUND: Although extensive research has been performed to control differentiation of neural stem cells - still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation - allowing for an increase in percentage yield of neuronal cells. RESULTS: Uncommitted GFAP and SOX2 positive neural progenitors - so-called, Normal Human Astrocytes (NHA) were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100beta-, SOX2-], or mixture of neural and non-neural cells.In spite of successfully increasing the percentage yield of glial and neuronal vs. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF) did not radically change the ratio between neuronal and glial cells--i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively. CONCLUSION: We suggest that biotechnologists attempting to enrich in vitro neural cell cultures in one type of cells - such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.

Neuronal and astrocytic cells, obtained after differentiation of human neural GFAP-positive progenitors, present heterogeneous expression of PrPc.

PrP(c) is a cellular isoform of the prion protein with an unknown normal function. One of the putative physiological roles of this protein is its involvement in cell differentiation. Recently, in vitro and in vivo studies showed that GFAP-positive cells have characteristics of stem/progenitor cells that generate neurons and glia. We used an in vitro model of human neurogenesis from GFAP-positive progenitor cells to study the expression of PrP(c) during neural differentiation. Semi-quantitative multiplex-PCR assay and Western blot analysis revealed a significant increase of PRNP expression level in differentiated cells compared to undifferentiated cell population. As determined by immunocytochemistry followed by a quantitative image analysis, the PrP(c) level increased significantly in neuronal cells and did not increase significantly in glial cells. Of note, glial and neuronal cells showed a very large heterogeneity of PrP(c) expression. Our results provide the basis for studying the role of PrP(c) in cell differentiation and neurogenesis from human GFAP-positive progenitor cells.

KCTD11 expression in medulloblastoma is lower than in adult cerebellum and higher than in neural stem cells.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood, and the most frequent associated genetic alteration is loss of heterozygosity on chromosome region 7p13. Two genes mapping to this region, KCTD11 (alias REN) and HIC1, have been proposed as involved in MB pathogenesis. We used real-time polymerase chain reaction in 20 tissue samples of primary MB to examine the transcriptional level of the two genes, with reference to two types of controls: adult cerebellum and fetal neural stem cells. A significant reduction of KCTD11 expression relative to adult normal cerebellum was detected in 14 of 20 (70%) of MB samples. Neural stem cells had even lower levels of KCTD11 expression than did MB. HIC1 gene expression was low ( approximately 100 times lower than KCTD11 expression) in MB, and low also in both adult cerebellum and neural stem cells. Hypermethylation of the 5'UTR or the central region of HIC1 (or both) was detected in a significant number of MB samples, as well as in cerebellum and neural stem cells. Our data suggest that KCTD11 may play an important role in MB tumorigenesis, but do not support the role of HIC1 in this tumor development. We argue that recognition of the gene or genes important in MB tumorigenesis depends in part on defining an appropriate control.

Regulation of PrPC expression: nerve growth factor (NGF) activates the prion gene promoter through the MEK1 pathway in PC12 cells.

A high expression of PrP(C) in cells is one factor that increases the risk of conversion to the misfolded, disease-associated form (PrP(Sc)) characteristic of transmissible spongiform encephalopathies. Thus, developing a method to control the level of PrP(C) expression in cells could be one way to delay or prevent the onset of clinical signs of these diseases. In this study the mechanisms controlling the expression of the Prnp gene in PC12 cells and in rat brain were examined. We observed a slight activation of a cloned fragment of the human PRNP gene promoter using the luciferase reporter system in PC12 cells stimulated with nerve growth factor (NGF). The activating effect of NGF was enhanced by mitogen-activated protein kinase (MEK1) and suppressed by myristylated serine/threonine kinase (myrAKT). These results suggest that MEK1 is a positive activator of the PRNP promoter that inhibits the AKT pathway. Independent experiments suggested that high expression of PrP(C) in the brain depends on the rate of translation and/or the efficiency of PrP(C) stabilization. We also investigated the epigenic status of the Prnp promoter. We observed no increase of PrP(C) or Prnp mRNA levels in PC12 cells after treatment with the DNA-demethylating agent. The Prnp promoter did not display methylation either in NGF-treated and untreated PC12 cells, or in the rat brain. These results improve the understanding of the regulation of the Prnp gene promoter, a DNA regulatory element controlling the expression of PrP(C), a protein involved in several neurological diseases.