Regulation of PTEN expression in neuronal apoptosis.

PTEN phosphatase is a tumor suppressor gene that dephosphorylates phosphatidylinositol phosphates. PTEN restrains the function of a major antiapoptotic and survival pathway involving phosphoinositide 3-kinase and Akt kinase. Our purpose was to find out whether apoptotic inducers affect the expression of PTEN in cerebellar granule neurons and neuroblastoma 2a cells (Neuro-2a). PTEN mRNA expression showed a major 5.5-kb and a lower abundance 2.5-kb transcripts. In Neuro-2a cells, serum withdrawal induced a prominent, continuous decrease both in 5.5- and 2.5-kb transcripts of PTEN mRNA. Simultaneously, the expression level of 56-kDa PTEN protein decreased in Neuro-2a cells. The decrease in PTEN expression precedes apoptotic changes observed after serum withdrawal. On the contrary, okadaic acid and etoposide only slightly affected the expression of PTEN although they induce a prominent apoptosis in Neuro-2a cells. In cerebellar granule neurons, okadaic acid treatment induced a prominent increase in PTEN mRNA expression after 6-h treatment, both at the 5.5- and 2.5-kb transcripts. The early response in PTEN mRNA expression disappeared in 5.5-kb transcripts already at 12 h and in the case of 2.5-kb transcripts it lasted up to 24 h. Potassium deprivation, known to induce apoptosis in cerebellar granule cells, did not affect PTEN mRNA expression but together with serum deprivation induced a clear decrease in the 5. 5-kb PTEN transcripts. It seems that the changes in PTEN expression level and neuronal apoptosis are not related to each other in general but the expression of PTEN phosphatase seems to regulate certain apoptotic signals affecting phosphoinositide 3-kinase function.

A differential display protocol to identify differentially expressed mRNAs in potassium-deprived cerebellar granule cells.

Differential display (DD) has become a popular technique for the identification of differentially expressed genes. Here we present a DD protocol for studying mRNA expression changes during neuronal apoptosis. Neuronal apoptosis is typically dependent on macromolecular synthesis, thus suggesting that regulation of gene expression is involved in control of the activation of the cell-death machinery. In order to identify some of the genes involved, we employed the widely used cell culture model in which apoptosis is induced in rat cerebellar granule cells (CGCs) using potassium deprivation. Although DD has been applied productively in the study of various biological phenomena, the method has its drawbacks. In particular, the cloning and verification of cDNA fragments is frequently described as problematic or laborious, and often produces many "false positives". Here we report the successful use of DD including an efficient protocol for cDNA clone screening and verification. This protocol avoids some of the problems presented by heterogeneous DD bands, which may be a major cause of false-positive results. To identify the desired clones, we apply single-stranded conformational polymorphism (SSCP) and slot blot techniques.

Assessment of Magical Beliefs about Food and Health.

The Magical Beliefs About Food and Health scale (MFH) was developed to assess individual differences in the tendency to adopt eating and health instructions that many magazines, health care books and food ideologies regard as valid but which obey universal laws of similarity and contagion. In a study of 216 individuals, the total MFH score showed good internal consistency and it was associated with various validity criteria as hypothesized (e.g. vegetarianism and other ideological commitments to food choice, female gender, increased neuroticism, experiential thinking, positive attitudes towards alternative medicine, low sensation seeking and endorsement of universalism values). Factor analysis yielded two factors: General Magical Beliefs and Animal Products as Food Contaminants. In addition, three other items (the Animal Products as Personality Contaminants scale) cross-loaded on the two factors. The factor structure and test-retest reliability were confirmed with separate samples. The results showed that the total MFH score is a reliable and valid measure of magical food and health beliefs, and that the subscales may prove useful when a multidimensional assessment of magical beliefs is needed.

Expression of seizure-related PTZ-17 is induced by potassium deprivation in cerebellar granule cells.

The aim of this study was to identify changes in gene expression during neuronal apoptosis using the differential display (DD) technique. Potassium deprivation was used to induce neuronal apoptosis in cultured rat cerebellar granule cells. DD analysis of about 1600 transcripts resulted in 8 cDNA clones that confirmed differential expression in a slot blot analysis. One of these clones was homologous to the 3' end of seizure-related PTZ-17 RNA. Northern blot analysis showed a marked upregulation of a 2.2 kb RNA 24 hours after potassium withdrawal. This upregulation was prevented by the RNA synthesis inhibitor actinomycin D. The increase in PTZ-17 expression was specific for potassium deprivation induced apoptosis, since the other apoptosis inducers, okadaic acid and staurosporine, did not affect PTZ-17 expression. The level of PTZ-17 RNA was not significantly affected by aging in rat cerebellum. Our data suggest that the upregulation of the PTZ-17 RNA is a part of the steps leading to apoptosis during potassium deprivation in cerebellar granule cells.

Insulin-like growth factor binding protein 5 and type-1 insulin-like growth factor receptor are differentially regulated during apoptosis in cerebellar granule cells.

Neuronal apoptosis is considered to play a significant role in several neuropathological conditions. However, the molecular mechanisms underlying neuronal apoptosis are poorly understood. Insulin-like growth factor (IGF) signalling is considered to be an important regulator of neuronal differentiation, survival and apoptosis. We have examined the expression of two members of the IGF system, insulin-like growth factor binding protein 5 (IGFBP-5) and the type-1 IGF receptor (IGF1R), during apoptosis of rat cerebellar granule cells (CGCs) in vitro. We describe a prominent downregulation of IGFBP-5 mRNA and protein expression. We also show that IGF-I increases IGFBP-5 expression in CGCs and that the downregulation of IGFBP-5 mRNA can be suppressed by inhibiting mRNA synthesis with actinomycin D. The expression of IGF1R mRNA showed a transient upregulation during potassium chloride (KCl) deprivation induced apoptosis, in contrast to the IGF1R protein level, which was downregulated during KCl deprivation. Our results provide insight into the expression of IGF-related genes during neuronal apoptosis, and indicate that they mediate a protective response to the withdrawal of trophic stimulation. It seems that the expression of IGFBP-5 and IGF1R is regulated to maximize the availability of IGF and the activity of IGF-triggered survival signalling.

Expression of transcriptional repressor proteins mSin3A and 3B during aging and replicative senescence.

Sin3 proteins have a key role in transcriptional repression mediated by histone deacetylation. Mammalian Sin3 proteins, mSin3A and 3B, act as adapter molecules which bind both to repressive transcription factors and to the methyl-CpG-binding proteins (MeCPs) and recruit histone deacetylases to assemble a multiprotein repressor complex. We have recently observed (Biochem. Biophys. Res. Commun. 252, 274-277, 1998) that the expression of mSin3A but not mSin3B protein is induced during neuronal apoptosis. The purpose of this study was to find out whether aging and replicative senescence affect the expression levels of mSin3A and 3B repressor proteins. We studied the expression levels of mSin3A and 3B mRNAs and proteins both in replicative senescence model of WI-38 fibroblasts and in liver and brain tissues of young (4-6 months) and old (26-30 months) male Wistar rats. Replicative senescence of human WI-38 fibroblasts did not affect the expression levels of mSin3A and 3B mRNAs. However, the late passage WI-38 fibroblasts showed a significant decline in the expression level of mSin3A protein. Immortalization of WI-38 fibroblasts with SV-40 transformation increased the expression level of 6.0 kb mSin3A mRNA. Aging of Wistar rats did not affect the expression levels of either mSin3A or mSin3B mRNAs in the liver and frontal cortex. Similarly, the protein levels of mSin3A and 3B were unaffected in the hippocampus, cerebellum and liver tissues during aging. These results show that aging in vivo, in contrast to replicative senescence, does not affect the expression levels of mSin3A and 3B repressor proteins. However, this does not exclude the possible age-related functional changes mediated by mSin3-histone deacetylase complexes.