Genome-scan for bipolar disorder with sib-pair families in the Sardinian population: a new susceptibility locus on chromosome 1p22-p21?

The discovery of the genetic factors implicated in the predisposition to complex diseases may greatly profit from genetic studies in isolated populations. In this perspective, we performed a genome-wide scan using 507 microsatellite markers, with an average interval size of 7.6 cM, on a sample of 88 nuclear families with at least two affected sibs with bipolar disorder recruited in the Sardinian population. An initial analysis yielded non-parametric linkage exceeding 3.4 with P-values <0.0003 at two adjacent markers, D1S206 and D1S435 in the 1p22-p21 chromosomal region. Moreover, positive linkage ranging between 2.0 and 3.0 was obtained for other loci in several cases in regions that have already been linked to predisposition to bipolar disorder, such as 5p15.33, 8q24.13, and 11q14.3. A subsequent analysis of the 1p22-p21 region using the same set of families and a dense panel of 20 new microsatellite markers, spaced at 1.2 cM on average, reinforced the finding of suggestive linkage for this region. Interestingly, NPL values above 2.1 and P-values <0.02 were obtained for a cluster of 10 markers comprising D1S435. Thus, this study suggests that the 1p22-p21 region may contain a new locus participating to the genetic susceptibility to bipolar disorder and reproduces positive linkage for several other loci already implicated in this pathology. Since the Sardinian population presents a peculiar genetic homogeneity, these results may pave the way to further studies for replication in this population contributing to the rapid discovery of the genetic factors predisposing to bipolar disorder.

The extract of Ginkgo biloba EGb 761 reactivates a juvenile profile in the skeletal muscle of sarcopenic rats by transcriptional reprogramming.

BACKGROUND: Sarcopenia is a major public health problem in industrialized nations, placing an increasing burden on public healthcare systems because the loss of skeletal muscle mass and strength that characterizes this affection increases the dependence and the risk of injury caused by sudden falls in elderly people. Albeit exercise and caloric restriction improve sarcopenia-associated decline of the muscular performances, a more suitable and focused pharmacological treatment is still lacking. METHODOLOGY/PRINCIPAL FINDINGS: In order to evaluate such a possible treatment, we investigated the effects of EGb 761, a Ginkgo biloba extract used in chronic age-dependent neurological disorders, on the function of the soleus muscle in aged rats. EGb 761 induced a gain in muscular mass that was associated with an improvement of the muscular performances as assessed by biochemical and electrophysiological tests. DNA microarray analysis shows that these modifications are accompanied by the transcriptional reprogramming of genes related to myogenesis through the TGFbeta signaling pathway and to energy production via fatty acids and glucose oxidation. EGb 761 restored a more juvenile gene expression pattern by regenerating the aged muscle and reversing the age-related metabolic shift from lipids to glucose utilization. CONCLUSIONS/SIGNIFICANCE: Thus, EGb 761 may represent a novel treatment for sarcopenia both more manageable and less cumbersome than exercise and caloric restriction.

Neonatal handling prevents the effects of phencyclidine in an animal model of negative symptoms of schizophrenia.

BACKGROUND: Environmental factors during the neonatal period have long-lasting effects on the brain. Neonatal handling, an early mild stress, enhances the ability to cope with stress in adult rats. In humans, inappropriate stress responses increase the risk of schizophrenia in genetically predisposed individuals. We studied the effect of neonatal handling on the phencyclidine (PCP)-induced immobility time of rats in the forced swimming test (FST, an animal model of negative symptoms of schizophrenia) and on plasma adrenocorticotropic hormone (ACTH) as a measure of hypothalamic-pituitary-adrenal axis (HPA) reactivity. METHODS: Pups were removed from their mothers 15 min/21 days after birth. Postnatal day 65: animals were submitted to restraint stress. Postnatal day 75: after PCP treatment (5 mg/kg/5 days) animals were submitted to the FST. RESULTS: Neonatal handling reduced HPA reactivity to passive stress (restraint) but not to active coping stress (forced swimming). Immobilization time was significantly lower in saline- and PCP-treated, handled animals than in non-handled ones. Handling prevented the ACTH increase induced by PCP that was observed in the non-handled rats after FST. CONCLUSIONS: First, neonatal handling protects animals from acquiring the schizophrenic-like behavior provoked by sub-chronic PCP treatment, which was associated with a reduced HPA activity. Second, the beneficial properties of handling in stress responses seem to depend on the type of stress.

Gi/Go protein-dependent presynaptic mechanisms are involved in clozapine-induced down-regulation of tyrosine hydroxylase in PC12 cells.

Although the clinical effects of antipsychotics have been extensively studied, the molecular mechanisms underlying their antipsychotic activity are unclear. Chronic clozapine has been reported to reduce significantly the expression of tyrosine hydroxylase (TH) in the mesolimbic system. To characterize the mechanisms of action of clozapine on TH expression, PC12 cells turned out to be a useful model, being by far less complex than the entire brain. Both the quantity of TH protein and the amount of TH mRNA in PC12 cells were found to be decreased during incubation of the cells in the presence of clozapine. This decline was followed by a decrease in the enzymatic activity of TH. The effect of clozapine was blocked by preincubation with N-ethylmaleimide, a sulphydryl-alkylating reagent that interferes in Gi/o protein-mediated second messenger pathways. Clozapine may thus decrease TH expression by interacting with Gi/o protein-coupled receptors, such as D2 and 5HT1A. Knowledge of the molecular mechanisms underlying the clinical effects of established antipsychotics will promote the development of new and more efficient antipsychotic drugs.

The tissue-specific methylation of the human tyrosine hydroxylase gene reveals new regulatory elements in the first exon.

The methylation status of CpG dinucleotides located in or near regulatory elements affects gene expression. The CpG-rich sequence located outside the 5' promoter region of the human Tyrosine Hydroxylase (TH) gene appears to influence the functional effect of the adjacent intronic HUMTH01 microsatellite. In order to identify new regulatory elements in this region acting on gene expression, the methylation profile of the TH CpG island was investigated using the bisulfite sequencing method. The overall methylation level of this region is correlated to TH-expressing and non-expressing status in cell lines and DNA demethylation treatment with 5-azacytidine increased TH expression. Moreover, in a homogeneous background of methylated CpGs, a single CpG in the first exon of the gene is constantly either unmethylated or methylated in, respectively, TH-expressing or non-expressing cell lines, tissues and single cells. Further analysis ascertained that this CpG is contained in a sequence characterized by putative binding sites for the AP2, Sp1 and KAISO factors. Characterization of this sequence shows that these factors specifically bind their respective sites. Finally, the binding of KAISO, a transcriptional repressor, is conditioned by the methylation of this sequence, which may, thus, participate in the regulation of TH gene expression according to its methylation pattern.

DNA microarrays and pharmacogenomics.

The DNA microarrays have proven to be a state of the art technique for high throughput comprehensive analysis of thousand of genes in parallel. The application of a DNA microarray to compare normal and pathological cells, tissues or organs may allow, along with classical positional cloning techniques, to speed up the discovery of genes and gene pathways implicated in several diseases. This in turn will result in further application of the DNA microarray technique in the field of pharmacogenomics in order to characterize and validate new therapeutic targets, their mechanism of action, metabolic pathways and unwanted secondary effects. However, the lack of standardized criteria for the analysis and interpretation of the huge amount of data generated by DNA microarrays may hamper its utilization on a routine basis in the human health domain.

Transforming growth factor beta1 overexpression in the nigrostriatal system increases the dopaminergic deficit of MPTP mice.

Idiopathic Parkinson's disease (PD) is characterized by mesencephalic dopaminergic neuron cell death and striatal dopamine (DA) depletion. The factors involved in the pathogenesis of the disease are still unknown. Transforming growth factor beta1 (TGFbeta1) is increased in the striatum of patients with PD. However, the effect of this increase is not known. Here, we show that overexpression of TGFbeta1, by recombinant adenovirus TGFbeta1 gene transfer, in the mesostriatal system of an MPTP mouse model of PD decreased the number of mesencephalic dopaminergic neurons. This effect also involved more extensive DA depletion in the striatum. Striatal enkephalin mRNA levels are augmented, suggesting a decrease in dopaminergic transmission to the postsynaptic target. In the absence of MPTP, TGFbeta1 greatly decreased the number of dopaminergic neurons in the ventral mesencephalon of fully mature mice. These results show that an increase in TGFbeta1 levels aggravate the parkinsonian status of MPTP mice and may therefore be a risk factor for the development of PD.

Post-genomic era and gene discovery for psychiatric diseases: there is a new art of the trade? The example of the HUMTH01 microsatellite in the Tyrosine Hydroxylase gene.

The microsatellite HUMTH01, located in the first intron of the Tyrosine Hydroxylase (TH) gene (encoding the rate-limiting enzyme in the synthesis of catecholamines), is characterized by a TCAT repeated motif and has been used in genetic studies of neuropsychiatric and other complex diseases, in which catecholaminergic neurotransmission is implicated. After reporting a positive association between HUMTH01 and bipolar disorder as well as schizophrenia, the authors established that HUMTH01 alleles display the features of regulatory elements. Thereafter, they cloned two proteins (ZNF191 and HBP1), specifically binding to HUMTH01, and demonstrated that allelic variations of HUMTH01 have a quantitative silencing effect on TH gene expression in vitro, and correlate with quantitative and qualitative changes in the binding by ZNF191. The authors aim to characterize the transduction pathway impinging on the HUMTH01 microsatellite and establish its relevance for TH gene regulation in vivo. Since the TCAT repeated sequence is widespread throughout the genome, their approach may lead to the dissection of the mechanisms underlying the quantitative expression of several genes implicated in complex genetic traits, both normal and pathological. Thus, these investigations on the possible contribution and potential role of the HUMTH01 microsatellite in neuro-pathological conditions may represent an example of the different approaches needed to validate genetic targets in the "post-genomic era."

Molecular mechanisms of neuronal cell death: implications for nuclear factors responding to cAMP and phorbol esters.

Chronic treatment with calcium ionophore A23187 in NGF-differentiated cells results in cell death that is time- and concentration-dependent. Additionally, PC12 cells codifferentiated with NGF and dBcAMP become dependent on these factors for survival and undergo apoptosis when both factors are withdrawn. We show that in both cases there is a prolonged induction of c-Fos which correlates with cell death. Its continual activation in PC12 cells overexpressing c-FosER results in caspase-3 cleavage and rapid cell death. Specific phosphorylation of CREB/CREM(tau) transactivators or their binding to CRE of c-fos was observed. Our results indicate that prolonged c-Fos induction activates p53. There is increased nuclear localization of p53, p21 and Bax levels are induced in NGF/dBcAMP-deprived c-FosER cells, and dominant negative p53 inhibits cell death induced either by serum deprivation or by c-Fos. Overall these data implicate AP-1 as a nuclear target of signal transduction pathways which plays a role in the activation of apoptosis.