Elena Milanesi

Glucocorticoid-Related Molecular Signaling Pathways Regulating Hippocampal Neurogenesis

Stress and glucocorticoid hormones regulate hippocampal neurogenesis, but the molecular mechanisms underlying their effects are unknown. We, therefore, investigated the molecular signaling pathways mediating the effects of cortisol on proliferation, neuronal differentiation, and astrogliogenesis, in an immortalized human hippocampal progenitor cell line. In addition, we examined the molecular signaling pathways activated in the hippocampus of prenatally stressed rats, characterized by persistently elevated glucocorticoid levels in adulthood. In human hippocampal progenitor cells, we found that low concentrations of cortisol (100 nM) increased proliferation (+16%), decreased neurogenesis into microtubule-associated protein 2 (MAP2)-positive neurons (−24%) and doublecortin (Dcx)-positive neuroblasts (−21%), and increased differentiation into S100β-positive astrocytes (+23%). These effects were dependent on the mineralocorticoid receptor (MR) as they were abolished by the MR antagonist, spironolactone, and mimicked by the MR-agonist, aldosterone. In contrast, high concentrations of cortisol (100 μM) decreased proliferation (−17%) and neuronal differentiation into MAP2-positive neurons (−22%) and into Dcx-positive neuroblasts (−27%), without regulating astrogliogenesis. These effects were dependent on the glucocorticoid receptor (GR), blocked by the GR antagonist RU486, and mimicked by the GR-agonist, dexamethasone. Gene expression microarray and pathway analysis showed that the low concentration of cortisol enhances Notch/Hes-signaling, the high concentration inhibits TGFβ-SMAD2/3-signaling, and both concentrations inhibit Hedgehog signaling. Mechanistically, we show that reduced Hedgehog signaling indeed critically contributes to the cortisol-induced reduction in neuronal differentiation. Accordingly, TGFβ-SMAD2/3 and Hedgehog signaling were also inhibited in the hippocampus of adult prenatally stressed rats with high glucocorticoid levels. In conclusion, our data demonstrate novel molecular signaling pathways that are regulated by glucocorticoids in vitro, in human hippocampal progenitor cells, and by stress in vivo, in the rat hippocampus.

Reduced peripheral brain-derived neurotrophic factor mRNA levels are normalized by antidepressant treatment

Consistent data coming from biochemical studies have evidenced a brain-derived neurotrophic factor (BDNF) serum reduction in depressed patients compared to controls and a restoration following antidepressant treatment. However, to date, no study has evaluated whether BDNF synthesis in leukocytes could contribute to such modulation. Therefore, in this study, we analysed BDNF mRNA levels in leukocytes from 21 depressed patients prior to and during escitalopram treatment and from 23 control subjects showing that BDNF mRNA levels were decreased in drug-free depressed patients and that 12 wk escitalopram treatment was able to reverse this deficit. Interestingly, changes in BDNF mRNA levels paralleled BDNF serum increase during antidepressant treatment, and were correlated with symptoms improvement. Our results indicate that BDNF serum modulation observed in depressed patients is associated with BDNF synthesis alteration in leukocytes and suggest that these peripheral cells might play an active role in the mechanisms of action of antidepressants.

Altered Gene Expression in Schizophrenia: Findings from Transcriptional Signatures in Fibroblasts and Blood

Background Whole-genome expression studies in the peripheral tissues of patients affected by schizophrenia (SCZ) can provide new insight into the molecular basis of the disorder and innovative biomarkers that may be of great utility in clinical practice. Recent evidence suggests that skin fibroblasts could represent a non-neural peripheral model useful for investigating molecular alterations in psychiatric disorders. Methods A microarray expression study was conducted comparing skin fibroblast transcriptomic profiles from 20 SCZ patients and 20 controls. All genes strongly differentially expressed were validated by real-time quantitative PCR (RT-qPCR) in fibroblasts and analyzed in a sample of peripheral blood cell (PBC) RNA from patients (n = 25) and controls (n = 22). To evaluate the specificity for SCZ, alterations in gene expression were tested in additional samples of fibroblasts and PBCs RNA from Major Depressive Disorder (MDD) (n = 16; n = 21, respectively) and Bipolar Disorder (BD) patients (n = 15; n = 20, respectively). Results Six genes (JUN, HIST2H2BE, FOSB, FOS, EGR1, TCF4) were significantly upregulated in SCZ compared to control fibroblasts. In blood, an increase in expression levels was confirmed only for EGR1, whereas JUN was downregulated; no significant differences were observed for the other genes. EGR1 upregulation was specific for SCZ compared to MDD and BD. Conclusions Our study reports the upregulation of JUN, HIST2H2BE, FOSB, FOS, EGR1 and TCF4 in the fibroblasts of SCZ patients. A significant alteration in EGR1 expression is also present in SCZ PBCs compared to controls and to MDD and BD patients, suggesting that this gene could be a specific biomarker helpful in the differential diagnosis of major psychoses.

Copy number variants in attention-deficit hyperactive disorder: identification of the 15q13 deletion and its functional role.

Objectives Evidence has supported a role for rare copy number variants in the etiology of attention-deficit hyperactivity disorder (ADHD), in particular, the region 15q13, which is also a hot spot for several neuropsychiatric disorders. This region spans several genes, but their role and the biological implications remain unclear. Methods We carried out, for the first time, an analysis of the 15q13 region in an Italian cohort of 117 ADHD patients and 77 controls using the MLPA method, confirmed by a genome single-nucleotide polymorphism array. In addition, we probed for downstream effects of the 15q13 deletions on gene expression by carrying out a transcriptomic analysis in blood. Results We found 15q13 deletions in two ADHD patients and identified 129 genes as significantly dysregulated in the blood of the two ADHD patients carrying 15q13 deletions compared with ADHD patients without 15q13 deletions. As expected, genes in the deleted region (KLF13, MTMR10) were downregulated in the two patients with deletions. Moreover, a pathway analysis identified apoptosis, oxidation reduction, and immune response as the mechanisms that were altered most significantly in the ADHD patients with 15q13 deletions. Interestingly, we showed that deletions in KLF13 and CHRNA7 influenced the expression of genes belonging to the same immune/inflammatory and oxidative stress signaling pathways. Conclusion Our findings are consistent with the presence of 15q13 deletions in Italian ADHD patients. More interestingly, we show that pathways related to immune/inflammatory response and oxidative stress signaling are affected by the deletion of KFL13 and CHRNA7. Because the phenotypic effects of 15q13 are pleiotropic, our findings suggest that there are shared biologic pathways among multiple neuropsychiatric conditions.

BDNF Val66Met polymorphism and protein levels in Amniotic Fluid

BackgroundBrain-Derived Neurotrophic Factor (BDNF) is a neurotrophin which plays survival- and growth-promoting activity in neuronal cells and it is involved in cellular plasticity mechanisms as it controls activity dependent synaptic transmission. A functional polymorphism (Val66Met) in the pro-region of BDNF, which affects the intracellular trafficking of proBDNF has been associated with memory and cognitive deficits as well as to an increased susceptibility for several psychiatric disorders especially those with a neurodevelopmental origin. To date, no study has evaluated the influence of the Val66Met polymorphism on BDNF levels in a peripheral system that may reflect fetal neurodevelopment. Therefore we investigated in amniotic fluids (AF) obtained from 139 healthy women during 15-17 week of pregnancy, BDNF protein levels in correlation with the Val66Met polymorphism.ResultsInterestingly we found a significant BDNF protein levels reduction in 55 Met carriers (Val/Met and Met/Met) (p = 0.002) as compared to 84 non carriers (Val/Val), and no effect of fetus gender, maternal age or gestation week on BDNF levels has been observed.ConclusionThese results, although explorative, indicate that during fetal life the Val66Met genotype might influences BDNF protein levels in AF supporting the involvement of this polymorphism in behavioral and functional brain individual differences in the adulthood.

The role of GRIK4 gene in treatment-resistant depression

Several lines of evidence implicate abnormalities in glutamatergic neural transmission in major depressive disorder (MDD) and treatment response. A high percentage of MDD patients do not respond adequately to antidepressants and are classified as having treatment-resistant depression (TRD). In this study we investigated five GRIK4 variants, previously associated with antidepressants response, in an Italian cohort of 247 MDD no-TRD and 380 TRD patients. We found an association between rs11218030 G allele and TRD. Moreover, significant associations between rs11218030 and rs1954787 and the presence of psychotic symptoms were observed. In conclusion, our data support the involvement of GRIK4 in TRD and in the risk of developing psychotic symptoms during depressive episodes.

ErbB3 mRNA leukocyte levels as a biomarker for major depressive disorder

BackgroundIn recent years, the identification of peripheral biomarkers that are associated with psychiatric diseases, such as Major Depressive Disorder (MDD), has become relevant because these biomarkers may improve the efficiency of the differential diagnosis process and indicate targets for new antidepressant drugs. Two recent candidate genes, ErbB3 and Fgfr1, are growth factors whose mRNA levels have been found to be altered in the leukocytes of patients that are affected by bipolar disorder in a depressive state. On this basis, the aim of the study was to determine if ErbB3 and Fgfr1 mRNA levels could be a biomarkers of MDD.MethodsWe measured by Real Time PCR ErbB3 and Fgfr1 mRNA expression levels in leukocytes of MDD patients compared with controls. Successively, to assess whether ErbB3 mRNA levels were influenced by previous antidepressant treatment we stratified our patients sample in two cohorts, comparing drug-naive versus drug-free patients. Moreover, we evaluated the levels of the transcript in MDD patients after 12 weeks of antidepressant treatment, and in prefrontal cortex of rats stressed and treated with an antidepressant drug of the same class.ResultsThese results showed that ErbB3 but not Fgfr1 mRNA levels were reduced in leukocytes of MDD patients compared to healthy subjects. Furthermore, ErbB3 levels were not affected by antidepressant treatment in either human or animal modelsConclusionsOur data suggest that ErbB3 might be considered as a biomarker for MDD and that its deficit may underlie the pathopsysiology of the disease and is not a consequence of treatment. Moreover the study supports the usefulness of leukocytes as a peripheral system for identifying biomarkers in psychiatric diseases.

MTHFR: Genetic variants, expression analysis and COMT interaction in major depressive disorder

BACKGROUND Methylenetetrahydrofolate reductase (MTHFR) genetic variations have been widely studied in major depressive disorder (MDD) and antidepressants outcome. An interaction with catechol-O-methyltransferase (COMT) has also been proved affecting depression. The aim of this study was to clarify the role of the most commonly studied single nucleotide polymorphisms (SNPs) of MTHFR gene in MDD and in treatment response mechanisms, along with the impact of the interaction with COMT. METHODS A total of 613 MDD patients, of whom 389 were classified as having treatment resistant depression (TRD), and 463 controls were enrolled. The A1298C, C677T and COMT Val158Met were genotyped. Genetic data were integrated with a transcriptional level analysis in peripheral blood cells (PBCs) and fibroblasts. RESULTS The A1298C CC homozygotes were more frequent in MDD patients compared to controls in women, increasing twice the genetic risk to develop depression. Moreover this genotype resulted in epistasis with COMT Met carriers in association with MDD. No significant effects were obtained concerning response to treatment. Transcriptional analyses highlighted a strong correlation between the mRNA levels of MTHFR in fibroblasts and COMT genotypes whereas no significant association with MDD was found. PBCs results revealed relevant influences of environmental factors. LIMITATION We did not measure folate and homocisteine levels. CONCLUSION This study showed the involvement of A1298C, Val158Met and their interaction in MDD. The transcriptional analyses supported the participation of COMT in the folate pathway, which partakes in the complex network of gene×gene and gene×environment interactions of MDD etiopathogenesis.

Grant Application Review: The Case of Transparency

Grant Application Review: The Case of Transparency Public funding agencies should be more transparent in awarding research grants to allow researchers and the public better insight into decision making.

Understanding phenotype variability in frontotemporal lobar degeneration due to granulin mutation

Phenotype in patients with granulin (GRN) mutations is unpredictable, ranging from behavioral variant frontotemporal dementia (bvFTD) to agrammatic variant of primary progressive aphasia (avPPA). To date the wide clinical variability of FTLD-GRN remains unexplained. The aim of the study was to identify genetic pathways differentiating phenotypic expression in patients carrying GRN mutations. Patients carrying the same GRNT272SfsX10 mutation were enrolled, a careful clinical assessment was carried out, and the diagnosis of either bvFTD (n = 10, age = 63.9 ± 9.4) or avPPA (n = 6, age = 58.8 ± 4.7) was done. Microarray gene expression analysis on leukocytes was performed. Genes differentially expressed between the groups were validated by real time polymerase chain reaction considering an age-matched healthy controls group (n = 16, age = 58.4 ± 10.7). We further considered a group of FTD with no GRN mutations (GRN-) (n = 21, 13 bvFTD, and 8 avPPA) for comparisons. Real-time polymerase chain reaction (PCR) confirmed a significant decrease in leukocytes mRNA messenger RNA (mRNA) levels of RAP1GAP in bvFTD patients as compared with avPPA (p = 0.049). This finding was specific for patients with GRN mutations, as we did not observe this pattern in FTD GRN-patients (p = 0.99). The alteration of RAP1GAP mRNA levels may explain the clinical variability of GRN-FTLD patients. This is the first report linking a molecular pathway to specific phenotype expression in FTLD-GRN. To understand the clinical relevance of our early results it will be mandatory to extend the observation to other clinical and neuropathological series.