FoxP1 marks medium spiny neurons from precursors to maturity and is required for their differentiation.

Identifying the steps involved in striatal development is important both for understanding the striatum in health and disease, and for generating protocols to differentiate striatal neurons for regenerative medicine. The most prominent neuronal subtype in the adult striatum is the medium spiny projection neuron (MSN), which constitutes more than 85% of all striatal neurons and classically expresses DARPP-32. Through a microarray study of genes expressed in the whole ganglionic eminence (WGE: the developing striatum) in the mouse, we identified the gene encoding the transcription factor Forkhead box protein P1 (FoxP1) as the most highly up-regulated gene, thus providing unbiased evidence for the association of FoxP1 with MSN development. We also describe the expression of FoxP1 in the human fetal brain over equivalent gestational stages. FoxP1 expression persisted through into adulthood in the mouse brain, where it co-localised with all striatal DARPP-32 positive projection neurons and a small population of DARPP-32 negative cells. There was no co-localisation of FoxP1 with any interneuron markers. FoxP1 was detectable in primary fetal striatal cells following dissection, culture, and transplantation into the adult lesioned striatum, demonstrating its utility as an MSN marker for transplantation studies. Furthermore, DARPP-32 expression was absent from FoxP1 knock-out mouse WGE differentiated in vitro, suggesting that FoxP1 is important for the development of DARPP-32-positive MSNs. In summary, we show that FoxP1 labels MSN precursors prior to the expression of DARPP-32 during normal development, and in addition suggest that FoxP1 labels a sub-population of MSNs that are not co-labelled by DARPP-32. We demonstrate the utility of FoxP1 to label MSNs in vitro and following neural transplantation, and show that FoxP1 is required for DARPP-32 positive MSN differentiation in vitro.

Prostate cancer, miRNAs, metallothioneins and resistance to cytostatic drugs.

MicroRNAs (miRNAs) translationally repressing their target messenger RNAs due to their gene-regulatory functions play an important but not unexpected role in a tumour development. More surprising are the findings that levels of various miRNAs are well correlated with presence of specific tumours and formation of metastases. Moreover, these small regulatory molecules play a role in the resistance of cancer cells to commonly used anti-cancer drugs, such as cisplatin, anthracyclines, and taxanes. In that respect, miRNAs become very attractive target for potential therapeutic interventions. Improvements in the sensitivity of miRNAs detection techniques led to discovery of circulating miRNAs which became very attractive non-invasive biomarker of cancer with a substantial predictive value. In this review, the authors focus on i) oncogenic and anti-tumour acting miRNAs, ii) function of miRNAs in tumour progression, iii) possible role of miRNAs in resistance to anticancer drugs, and iv) diagnostic potential of miRNAs for identification of cancer from circulating miRNAs with special emphasis on prostate cancer. Moreover, relationship between miRNAs and expression of metallothionein is discussed as a possible explanation of resistance against platinum based drugs.

Insertional mutagenesis of preneoplastic astrocytes by Moloney murine leukemia virus.

Retroviral infection can induce transcriptional activation of genes flanking the sites of proviral integration in target cells. Because integration is essentially random, this phenomenon can be exploited for random mutagenesis of the genome, and analysis of integration sites in tumors may identify potential oncogenes. Here we have investigated this strategy in the context of astrocytoma progression. Neuroectodermal explants from astrocytoma-prone GFAP-v-src transgenic mice were infected with the ecotropic Moloney murine leukemia virus (Mo-MuLV). In situ hybridization and FACS analysis indicated that astrocytes from E12.5-13.5 embryos were highly susceptible to retroviral infection and expressed viral RNA and proteins both in vitro and in vivo. In average 80% of neuroectodermal cells were infected in vitro with 9-14 proviral integrations per cell. Virus mobility assays confirmed that Mo-MuLV remained transcriptionally active and replicating in neuroectodermal primary cultures even after 45 days of cultivation. Proviral insertion sites were investigated by inverse long-range PCR. Analysis of a limited number of provirus flanking sequences in clones originated from in vitro infected GFAP-v-src neuroectodermal cells identified loci of possible relevance to tumorigenesis. Therefore, the approach described here might be suitable for acceleration of tumorigenesis in preneoplastic astrocytes. We expect this method to be useful for identifying genes involved in astrocytoma development/progression in animal models.

Neuroinvasion of prions: insights from mouse models.

The prion was defined by Stanley B. Prusiner as the infectious agent that causes transmissible spongiform encephalopathies. A pathological protein accumulating in the brain of scrapie-infected hamsters was isolated in 1982 and termed prion protein (PrPSc). Its cognate gene Prnp was identified more than a decade ago by Charles Weissmann, and shown to encode the host protein PrP(C). Since the latter discovery, transgenic mice have contributed many important insights into the field of prion biology, including the understanding of the molecular basis of the species barrier for prions. By disrupting the Prnp gene, it was shown that an organism that lacks PrP(C) is resistant to infection by prions. Introduction of mutant PrP genes into PrP-deficient mice was used to investigate the structure-activity relationship of the PrP gene with regard to scrapie susceptibility. Ectopic expression of PrP in PrP knockout mice proved a useful tool for the identification of host cells competent for prion replication. Finally, the availability of PrP knockout mice and transgenic mice overexpressing PrP allows selective reconstitution experiments aimed at expressing PrP in neurografts or in specific populations of haemato- and lymphopoietic cells. The latter studies have allowed us to clarify some of the mechanisms of prion spread and disease pathogenesis.

Prions: pathogenesis and reverse genetics.

Spongiform encephalopathies are a group of infectious neurodegenerative diseases. The infectious agent that causes transmissible spongiform encephalopathies was termed prion by Stanley Prusiner. The prion hypothesis states that the partially protease-resistant and detergent-insoluble prion protein (PrPsc) is identical with the infectious agent, and lacks any detectable nucleic acids. Since the latter discovery, transgenic mice have contributed many important insights into the field of prion biology. The prion protein (PrPc) is encoded by the Prnp gene, and disruption of Prnp leads to resistance to infection by prions. Introduction of mutant PrPc genes into PrPc-deficient mice was used to investigate structure-activity relationships of the PrPc gene with regard to scrapie susceptibility. Ectopic expression of PrPc in PrPc knockout mice proved a useful tool for the identification of host cells competent for prion replication. Finally, the availability of PrPc knockout and transgenic mice overexpressing PrPc allowed selective reconstitution experiments aimed at expressing PrPc in neurografts or in specific populations of hemato- and lymphopoietic cells. The latter studies helped in elucidating some of the mechanisms of prion spread and disease pathogenesis.

[Direct DNA diagnosis of Friedreich's ataxia]. / Prímá DNA diagnostika Friedreichovy ataxie.

BACKGROUND: Friedreich's ataxia is an autosomal recessive, neurodegenerative disease with a prevalence of 1-2: 100,000. Ninety five % of cases are caused by Friedreich's ataxia expansion of GAA triplet repeat in the first intron of the X25 gene. The gene is mapped on chromosome 9q. The objective of the investigation was to introduce simple and reliable DNA diagnosis helping to specify of spinocerebellare ataxias. METHODS AND RESULTS: Our diagnosis is based on the differentiation of normal and mutant alleles of gene X25 with PCR and electrophoresis on agarose gel. Size of PCR product of normal allele is in our case 521-614 bp. It is responding to 7-38 GAA triplets. Size of mutant alleles with 200-1200 GAA triplets is as 4100 bp. After the method was introduced, we analysed 12 probands. Four of them suffered from Friedreich's ataxia. CONCLUSIONS: We introduced a fast, non-radioactive, reliable DNA diagnostic method. The contribution of this method is defection of carriers and we can screen of families with the risk of Friedreich's ataxia.

Haplotype analysis of the fragile X syndrome gene FMR1 in the Czech Republic.

We report on the haplotype analysis with polymorphic repeat markers DXS548 and FRAXAC1 next to the FMR1 gene in 37 unrelated fragile X and 36 control chromosomes from Bohemia and Moravia. Our results suggest a significant linkage disequilibrium between fragile X mutations and certain DXS548-FRAXAC1 haplotypes. Allele frequencies obtained differ slightly from those of other European populations with allele 194 being less frequent in our control sample. Rare DXS548 alleles 6.5 (195) and 0 (208) were also present.

[Direct detection of mutations in the LDL receptor gene in patients with familial hypercholesterolemia]. / Prímá detekce mutací v genu pro LDL receptor u pacientu s familiární hypercholesterolémií.

BACKGROUND: Familial hypercholesterolemia is one of the most frequent hereditary metabolic diseases. As a result of the functional disorder of the molecule of the LDL receptor LDL cholesterol is not sufficiently eliminated from the blood stream and exerts an atherogenic effect. The objective of the study was to introduce direct detection of mutations in the gene for the LDL receptor and characterize the spectrum of mutations in the Czech population. METHODS AND RESULTS: The authors analyzed a group of 84 unrelated patients where on the basis of clinical and biochemical criteria the diagnosis of FH was established. From the group 12 patients were eliminated (14.3%) where a mutation 3500 in the gene for apolipoprotein (apo) B-100 was detected. This mutation is most frequently the cause of a familial defect of apo B-100 (FDB), which cannot be differentiated clinically or biochemically from FH. In the LDL receptor gene a total of 11 mutations were found in 14 unrelated patients (16.7%), incl. 7 mutations not described hitherto. CONCLUSIONS: This is the first systematic characteristic of the spectrum of mutations in the LDL receptor gene in the Czech population. Molecular genetic analysis of the gene for the LDL receptor in affected families can contribute towards early assessment of the diagnosis of FH and thus to prevention of life threatening cardiovascular episodes in asymptomatic subjects.

Characterization of two nonsense mutations in the human dystrophin gene.

Forty Duchenne muscular dystrophy patients from the province of Moravia in the Czech Republic, who were previously found negative for large deletions in the dystrophin gene, were tested for the presence of point mutations in selected exons. Besides several intron and exon polymorphisms, two cases of nonsense mutations were detected in exon 70, thus causing the loss of the C-terminal domain of dystrophin. One of these, the mutation, S3365X, is newly reported here while the other, R3381X, has been described previously. These mutations, only 16 bp distant from each other, have a very different impact on the mental abilities of the corresponding patients.