Massimo Gennarelli

A Genome-Wide Investigation of SNPs and CNVs in Schizophrenia

We report a genome-wide assessment of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) in schizophrenia. We investigated SNPs using 871 patients and 863 controls, following up the top hits in four independent cohorts comprising 1,460 patients and 12,995 controls, all of European origin. We found no genome-wide significant associations, nor could we provide support for any previously reported candidate gene or genome-wide associations. We went on to examine CNVs using a subset of 1,013 cases and 1,084 controls of European ancestry, and a further set of 60 cases and 64 controls of African ancestry. We found that eight cases and zero controls carried deletions greater than 2 Mb, of which two, at 8p22 and 16p13.11-p12.4, are newly reported here. A further evaluation of 1,378 controls identified no deletions greater than 2 Mb, suggesting a high prior probability of disease involvement when such deletions are observed in cases. We also provide further evidence for some smaller, previously reported, schizophrenia-associated CNVs, such as those in NRXN1 and APBA2. We could not provide strong support for the hypothesis that schizophrenia patients have a significantly greater “load” of large (>100 kb), rare CNVs, nor could we find common CNVs that associate with schizophrenia. Finally, we did not provide support for the suggestion that schizophrenia-associated CNVs may preferentially disrupt genes in neurodevelopmental pathways. Collectively, these analyses provide the first integrated study of SNPs and CNVs in schizophrenia and support the emerging view that rare deleterious variants may be more important in schizophrenia predisposition than common polymorphisms. While our analyses do not suggest that implicated CNVs impinge on particular key pathways, we do support the contribution of specific genomic regions in schizophrenia, presumably due to recurrent mutation. On balance, these data suggest that very few schizophrenia patients share identical genomic causation, potentially complicating efforts to personalize treatment regimens.

Serum and plasma BDNF levels in major depression: A replication study and meta-analyses

Abstract Objectives. Alterations of BDNF signalling in major depression (MD) are supported by studies demonstrating decreased levels of the neurotrophin serum and plasma content in MD patients. We conducted a replication study and we performed two meta-analyses on studies analysing serum and plasma BDNF levels in MD patients. Methods. The samples were composed by 489 patients/483 controls for the meta-analysis on serum and by 161 patients/211 controls for that on plasma levels. We performed also subgroup analyses to examine whether the decrease in BDNF levels in MD was influenced by gender. Results. In the replication study we found decreased serum BDNF levels in MD patients (P<0.01) and we demonstrated that is down-regulated the mature form of the neurotrophin (mBDNF). No significant difference was evidenced for plasma BDNF levels. The meta-analyses showed a reduction of both BDNF serum (P<0.0001) and plasma levels (P=0.02) in MD. No difference in the effect size on serum BDNF was observed between males and females (P=0.18). Conclusions. In conclusion, our results provide evidence of peripheral BDNF alteration in MD and support the rationale for further investigation aiming to the identification of biomarkers for differential diagnosis and personalization of therapies in this disorder.

Association between the BDNF 196 A/G polymorphism and sporadic Alzheimer's disease

SIR – Alzheimers disease (AD) is a chronic brain disorder associated with specific pathological changes resulting in neurodegeneration and in progressive development of dementia. This disease is clinically characterized by memory, reasoning and speech disorders and pathologically by the presence of senile plaques (SP), neurofibrillary tangles, and loss of syn-apses. 1 There are various hypotheses regarding an involvement of genetic factors in the development of AD. Mutations of genes encoding amyloid precursor protein, presenilin-1 and presenilin-2 cause familial AD, 2,3 and the ⑀4 allele of apolipoprotein E (APOE) gene gives susceptibility to familial and sporadic AD. 4 However, this genetic marker cannot explain the overall genetic susceptibility and additional/other genes may be involved in the development of AD. Genes involved in the neurodevelopmental process may be considered good candidates to confer susceptibility to AD. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family of growth factors which promotes survival, differentiation and maintenance of neurons in peripheral and central nervous system during normal development, 5 influences axonal growth and connectivity 6 and participates in the local responses to various types of neuronal stress or insults. 7 Table 1 Allele and genotype frequencies of the BDNF gene polymorphism at position 196 in Alzheimer patients and controls Alzheimer patients (n = 130) Healthy volunteers (n = 111) BDNF allele frequency Several lines of evidence have suggested altered functions of this gene in the pathogenesis of neurodeg-enerative diseases including Alzheimers disease: reduced levels of BDNF mRNA in the hippocampus, in the temporal cortex, in brain homogenates and in frontal cortex neurons have been found in individuals with AD. 8 In order to verify a potential role of the BDNF gene in the neuropathogenesis of Alzheimers disease, we analyzed allelic distribution of a polymorphism in the coding region of the BDNF gene in a diagnosed AD sample and in a control group. The polymorphism studied is a A/G (Met/Val) substitution located in the propeptide region, a highly unstable region constituted by 110 aa, in the BDNF gene at position 196 (codon 66) (SWISS.PROT: P23560.VAR 004626). Genomic DNA was extracted from blood samples obtained, after informed consent, from 130 Alzheimers patients (mean age 72 ± 3 years; 90 women and 40 men) recruited at the Alzheimer Unit of IRCCS-Fatebenefrat-elli (Brescia, Italy) and 111 healthy ethnically and age-matched volunteers. Patients and controls were Cauca-sians living in Northern Italy. Genotyping for this polymorphism has been done …

Candidate genes expression profile associated with antidepressants response in the GENDEP study: differentiating between baseline 'predictors' and longitudinal 'targets'.

To improve the ‘personalized-medicine’ approach to the treatment of depression, we need to identify biomarkers that, assessed before starting treatment, predict future response to antidepressants (‘predictors’), as well as biomarkers that are targeted by antidepressants and change longitudinally during the treatment (‘targets’). In this study, we tested the leukocyte mRNA expression levels of genes belonging to glucocorticoid receptor (GR) function (FKBP-4, FKBP-5, and GR), inflammation (interleukin (IL)-1α, IL-1β, IL-4, IL-6, IL-7, IL-8, IL-10, macrophage inhibiting factor (MIF), and tumor necrosis factor (TNF)-α), and neuroplasticity (brain-derived neurotrophic factor (BDNF), p11 and VGF), in healthy controls (n=34) and depressed patients (n=74), before and after 8 weeks of treatment with escitalopram or nortriptyline, as part of the Genome-based Therapeutic Drugs for Depression study. Non-responders had higher baseline mRNA levels of IL-1β (+33%), MIF (+48%), and TNF-α (+39%). Antidepressants reduced the levels of IL-1β (−6%) and MIF (−24%), and increased the levels of GR (+5%) and p11 (+8%), but these changes were not associated with treatment response. In contrast, successful antidepressant response was associated with a reduction in the levels of IL-6 (−9%) and of FKBP5 (−11%), and with an increase in the levels of BDNF (+48%) and VGF (+20%)—that is, response was associated with changes in genes that did not predict, at the baseline, the response. Our findings indicate a dissociation between ‘predictors’ and ‘targets’ of antidepressant responders. Indeed, while higher levels of proinflammatory cytokines predict lack of future response to antidepressants, changes in inflammation associated with antidepressant response are not reflected by all cytokines at the same time. In contrast, modulation of the GR complex and of neuroplasticity is needed to observe a therapeutic antidepressant effect.

Association between -G308A tumor necrosis factor alpha gene polymorphism and schizophrenia

Dysregulation of the inflammatory response system has been linked to pathophysiology of schizophrenia.1,2 Evidence of immune activation has derived from the detection of abnormal levels of proinflammatory cytokines and their receptors in peripheral blood and cerebrospinal fluid from schizophrenic patients.3–7 Cytokines are involved in normal CNS development as well as in the pathogenesis of many neuro-psychiatric disorders, acting directly on neural cells or modulating neurotransmitter and neuropeptide systems.8,9 In particular tumor necrosis factor α (TNFα), depending on its concentration, can exert both neurotrophic and neurotoxic effects and influence neural cell growth and proliferation.10,11 Moreover, TNFα gene is located on the small arm of chromosome 6 (6p21.1–21.3), a locus associated with genetic susceptibility to schizophrenia.12,13 We studied the distribution of −G308A TNFα gene polymorphism in 84 schizophrenic patients and in 138 healthy volunteers. This biallelic base exchange polymorphism directly affects TNFα plasma levels.14–18 Frequency of the TNF2(A) allele is significantly increased in schizophrenic patients as compared to controls (P = 0.0042). Genotype distribution is also significantly different (P = 0.0024). TNF2 homozygotes are represented only in the patient group (P = 0.002). These data suggest a potential role of TNFα as a candidate gene for susceptibility to schizophrenia and suggest that immune dysregulation in schizophrenic patients could also have a genetic component.

Mutation within TARDBP leads to Frontotemporal Dementia without motor neuron disease

It has been recently demonstrated that the 43‐kDa transactive response (TAR)‐DNA‐binding protein (TARDBP) is the neuropathological hallmark of Frontotemporal Dementia (FTD) with ubiquitin‐positive and tau‐negative inclusions. Large series of FTD patients without motor neuron disease have been previously analysed, but no TARDBP mutation was identified. The aim of the present study was to evaluate whether TARDBP gene mutations may be associated with FTD. We report that a pathogenetic TARDBP mutation is causative of behavioural variant FTD (bvFTD). An aged woman in her seventies initially started to present apathy and depression associated with impairment in executive functions. The diagnosis of bvFTD (apathetic syndrome) was accomplished by three‐year follow‐up, and structural and functional neuroimaging. By five‐years after onset, extensive electrophysiological investigations excluded subclinical motor neuron disease. In this patient, a single base substitution c.800A>G of TARDBP gene was identified. This mutation, already described as causative of ALS, predicted the amino acidic change arginine to serine at position 267 (N267S). In silico analysis demonstrated that this substitution generates a new phosphorylation site, and western blot analysis on lymphoblastoid cells reported a decrease of protein expression in N267S mutation carrier. Our study suggests that TARDBP mutations can be pathogenetic of bvFTD without motor neuron disease. TARDBP screening needs to be considered in FTD cases.

Selective phosphorylation of nuclear CREB by fluoxetine is linked to activation of CaM kinase IV and MAP kinase cascades

Regulation of gene expression is purported as a major component in the long-term action of antidepressants. The transcription factor cAMP-response element-binding protein (CREB) is activated by chronic antidepressant treatments, although a number of studies reported different effects on CREB, depending on drug types used and brain areas investigated. Furthermore, little is known as to what signaling cascades are responsible for CREB activation, although cAMP-protein kinase A (PKA) cascade was suggested to be a central player. We investigated how different drugs (fluoxetine (FLX), desipramine (DMI), reboxetine (RBX)) affect CREB expression and phosphorylation of Ser133 in the hippocampus and prefrontal/frontal cortex (PFCX). Acute treatments did not induce changes in these mechanisms. Chronic FLX increased nuclear phospho-CREB (pCREB) far more markedly than pronoradrenergic drugs, particularly in PFCX. We investigated the function of the main signaling cascades that were shown to phosphorylate and regulate CREB. PKA did not seem to account for the selective increase of pCREB induced by FLX. All drug treatments markedly increased the enzymatic activity of nuclear Ca2+/calmodulin (CaM) kinase IV (CaMKIV), a major neuronal CREB kinase, in PFCX. Activation of this kinase was due to increased phosphorylation of the activatory residue Thr196, with no major changes in the expression levels of α- and β-CaM kinase kinase, enzymes that phosphorylate CaMKIV. Again in PFCX, FLX selectively increased the expression level of MAP kinases Erk1/2, without affecting their phosphorylation. Our results show that FLX exerts a more marked effect on CREB phosphorylation and suggest that CaMKIV and MAP kinase cascades are involved in this effect.

Candidate Genes Expression Profile Associated with Antidepressants Response in the GENDEP Study

To improve the ‘personalized-medicine’ approach to the treatment of depression, we need to identify biomarkers that, assessed before starting treatment, predict future response to antidepressants (‘predictors’), as well as biomarkers that are targeted by antidepressants and change longitudinally during the treatment (‘targets’). In this study, we tested the leukocyte mRNA expression levels of genes belonging to glucocorticoid receptor (GR) function (FKBP-4, FKBP-5, and GR), inflammation (interleukin (IL)-1α, IL-1β, IL-4, IL-6, IL-7, IL-8, IL-10, macrophage inhibiting factor (MIF), and tumor necrosis factor (TNF)-α), and neuroplasticity (brain-derived neurotrophic factor (BDNF), p11 and VGF), in healthy controls (n=34) and depressed patients (n=74), before and after 8 weeks of treatment with escitalopram or nortriptyline, as part of the Genome-based Therapeutic Drugs for Depression study. Non-responders had higher baseline mRNA levels of IL-1β (+33%), MIF (+48%), and TNF-α (+39%). Antidepressants reduced the levels of IL-1β (−6%) and MIF (−24%), and increased the levels of GR (+5%) and p11 (+8%), but these changes were not associated with treatment response. In contrast, successful antidepressant response was associated with a reduction in the levels of IL-6 (−9%) and of FKBP5 (−11%), and with an increase in the levels of BDNF (+48%) and VGF (+20%)—that is, response was associated with changes in genes that did not predict, at the baseline, the response. Our findings indicate a dissociation between ‘predictors’ and ‘targets’ of antidepressant responders. Indeed, while higher levels of proinflammatory cytokines predict lack of future response to antidepressants, changes in inflammation associated with antidepressant response are not reflected by all cytokines at the same time. In contrast, modulation of the GR complex and of neuroplasticity is needed to observe a therapeutic antidepressant effect.

Electroconvulsive Therapy (ECT) increases serum Brain Derived Neurotrophic Factor (BDNF) in drug resistant depressed patients

Several findings have suggested that the neurotrophin BDNF could contribute to clinical efficacy of antidepressant treatments. The purpose of this study was to analyse if ECT operates a modulation of serum BDNF levels in a sample of drug resistant depressed patients. The results obtained show significantly higher serum levels of BDNF following ECT. More specifically, while no change occurred in the whole sample between T0 (baseline) and T1 (after ECT) (p=0.543) a significant increase has been identified at T2, one month after the end of ECT (p=0.002). However, the BDNF augmentation was evident even between T0 and T1 in a subgroup of patients who has low baseline BDNF levels. Although future researches are needed, the results herein presented show for the first time that ECT is associated with changes in serum BDNF and further support the possible involvement of BDNF in antidepressant therapies.

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.