Antonina Sidoti

The molecular interaction between the glutamatergic, noradrenergic, dopaminergic and serotoninergic systems informs a detailed genetic perspective on depressive phenotypes.

The glutamatergic pathway has been consistently involved in the physiopathology of depressive disorder. However a complete dissection and integration of its role in the context of other known mechanisms is lacking. We summarized and integrated the evidence of various levels of interaction between glutamatergic and monoaminergic pathways (see videos). We identified six molecular pathways, some of which with specific regional distribution within the brain. From the six pathways we identified the key proteins and their coding genes, we then provided a detailed list of possible candidates with practical suggestions for association studies planning.

Mutation Analysis of CCM1, CCM2 and CCM3 Genes in a Cohort of Italian Patients with Cerebral Cavernous Malformation

Cerebral cavernous malformations (CCMs) are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities. CCMs can occur as sporadic or familial autosomal dominant form. Familial cases are associated with mutations in CCM1[K‐Rev interaction trapped 1 (KRIT1)], CCM2 (MGC4607) and CCM3 (PDCD10) genes. In this study, a three‐gene mutation screening was performed by direct exon sequencing, in a cohort of 95 Italian patients either sporadic or familial, as well as on their at‐risk relatives. Sixteen mutations in 16 unrelated CCM patients were identified, nine mutations are novel: c.413T > C; c.601C > T; c.846 + 2T > G; c.1254delA; c.1255‐4delGTA; c.1681‐1682delTA in CCM1; c.48A > G; c.82‐83insAG in CCM2; and c.396G > A in CCM3 genes. The samples, negative to direct exon sequencing, were investigated by MLPA to search for intragenic deletions or duplications. One deletion in CCM1 exon 18 was detected in a sporadic patient. Among familial cases 67% had a mutation in CCM1, 5.5% in CCM2, and 5.5% in CCM3, whereas in the remaining 22% no mutations were detected, suggesting the existence of either undetectable mutations or other CCM genes. This study represents the first extensive research program for a comprehensive molecular screening of the three known genes in an Italian cohort of CCM patients and their at‐risk relatives.

Distribution and Localization of Vinculin-Talin-Integrin System and Dystrophin-Glycoprotein Complex in Human Skeletal Muscle

The vinculin-talin-integrin system and the dystrophin-glycoprotein complex (DGC) are two protein systems with structural and signaling functions, allowing interaction between muscle fibers and extracellular matrix. Although numerous studies have been conducted on these systems, their localization and distribution patterns along the nonjunctional sarcolemma are not clear. On this basis, we carried out an indirect immunofluorescence study on the vastus lateralis muscle of human adults not affected by neuromuscular diseases to better define these patterns. Our results showed that all tested proteins of the two systems have a costameric distribution; all tested proteins of the two systems colocalize with each other (about 90–95% of the cases); only α-sarcoglycan in a few cases (about 6%) does not colocalize with other proteins; in about 9–10% of the cases, dystrophin and β-dystroglycan colocalize partially with other proteins; all tested proteins can be localized in different fibers, both in the region of the sarcolemma over I or A bands. The colocalization between the vinculin-talin-integrin and DGC systems may imply their functional interaction involving the structural aspect, by providing a stronger adhesion between sarcolemma and extracellular matrix in well-defined regions of the muscle fiber. Besides, their colocalization may suggest the existence of a mechanism of mutual modulation of the transmitted signals. This reciprocal control may determine, in different conditions, the prevalence of one system over another with a consequent transmission of different messages to the sarcolemma-associated cytoskeleton.

Costameric proteins in human skeletal muscle during muscular inactivity.

Costameres are regions that are associated with the sarcolemma of skeletal muscle fibres and comprise proteins of the dystrophin–glycoprotein complex and vinculin–talin–integrin system. Costameres play both a mechanical and a signalling role, transmitting force from the contractile apparatus to the extracellular matrix in order to stabilize skeletal muscle fibres during contraction and relaxation. Recently, it was shown that bidirectional signalling occurs between sarcoglycans and integrins, with muscle agrin potentially interacting with both types of protein to enable signal transmission. Although numerous studies have been carried out on skeletal muscle diseases, such as Duchenne muscular dystrophy, recessive autosomal muscular dystrophies and other skeletal myopathies, insufficient data exist on the relationship between costameres and the pathology of the second motor nerve and between costameric proteins and muscle agrin in other conditions in which skeletal muscle atrophy occurs. Previously, we carried out a preliminary study on skeletal muscle from patients with sensitive‐motor polyneuropathy, in which we analysed the distribution of sarcoglycans, integrins and agrin by immunostaining only. In the present study, we have examined the skeletal muscle fibres of ten patients with sensitive‐motor polyneuropathy. We used immunofluorescence and reverse transcriptase PCR to examine the distribution of vinculin, talin and dystrophin, in addition to that of those proteins previously studied. Our aim was to characterize in greater detail the distribution and expression of costameric proteins and muscle agrin during this disease. In addition, we used transmission electron microscopy to evaluate the structural damage of the muscle fibres. The results showed that immunostaining of α7B‐integrin, β1D‐integrin and muscle agrin appeared to be severely reduced, or almost absent, in the muscle fibres of the diseased patients, whereas staining of α7A‐integrin appeared normal, or slightly increased, compared with that in normal skeletal muscle fibres. We also observed a lower level of α7B‐ and β1D‐integrin mRNA and a normal, or slightly higher than normal, level of α7A‐integrin mRNA in the skeletal muscle fibres of the patients with sensitive‐motor polyneuropathy, compared with those in the skeletal muscle of normal patients. Additionally, transmission electron microscopy of transverse sections of skeletal muscle fibres indicated that the normal muscle fibre architecture was disrupted, with no myosin present inside the actin hexagons. Based on our results, we hypothesize that skeletal muscle inactivity, such as that found after denervation, could result in a reorganization of the costameres, with α7B‐integrin being replaced by α7A‐integrin. In this way, the viability of the skeletal muscle fibre is maintained. It will be interesting to clarify, by future experimentation, the mechanisms that lead to the down‐regulation of integrins and agrin in muscular dystrophies.

Sarcoglycan Subcomplex Expression in Normal Human Smooth Muscle

The sarcoglycan complex (SGC) is a multimember transmembrane complex interacting with other members of the dystrophin–glycoprotein complex (DGC) to provide a mechanosignaling connection from the cytoskeleton to the extracellular matrix. The SGC consists of four proteins (α, β, γ, and δ). A fifth sarcoglycan subunit, ∊-sarcoglycan, shows a wider tissue distribution. Recently, a novel sarcoglycan, the ζ-sarcoglycan, has been identified. All reports about the structure of SGC showed a common assumption of a tetrameric arrangement of sarcoglycans. Addressing this issue, our immunofluorescence and molecular results showed, for the first time, that all sarcoglycans are always detectable in all observed samples. Therefore, one intriguing possibility is the existence of a pentameric or hexameric complex considering ζ-sarcoglycan of SGC, which could present a higher or lower expression of a single sarcoglycan in conformity with muscle type—skeletal, cardiac, or smooth—or also in conformity with the origin of smooth muscle. (J Histochem Cytochem 55:831–843, 2007)

Evaluation of the role of MAPK1 and CREB1 polymorphisms on treatment resistance, response and remission in mood disorder patients

Treatment resistant depression (TRD) is a significant clinical and public health problem. Among others, neuroplasticity and inflammatory pathways seem to play a crucial role in the pathomechanisms of antidepressant efficacy. The primary aim of this study was to investigate whether a set of single nucleotide polymorphisms (SNPs) within two genes implicated in neuroplasticity and inflammatory processes (the mitogen activated protein kinase 1, MAPK1 (rs3810608, rs6928, rs13515 and rs8136867), and the cyclic AMP responsive element binding protein 1, CREB1 (rs889895, rs6740584, rs2551922 and rs2254137)) was associated with antidepressant treatment resistance (according to two different definitions), in 285 Major Depressive Disorder (MDD) patients. As secondary aims, we investigated the genetic modulation of the same SNPs on response, remission and other clinical features both in MDD patients and in a larger sample including 82 Bipolar Disorder (BD) patients as well. All patients were screened in the context of a European multicenter project. No association between both the investigated genes and treatment resistance and response was found in MDD patients. However, considering remission, higher rates of CREB1 rs889895 GG genotype were reported in MDD patients. Moreover, MAPK1 rs8136867 AG genotype was found to be associated with remission in the whole sample (MDD and BD). Present results suggest that some genetic polymorphisms in both CREB1 and MAPK1 could be associated with treatment remission. Although further research is needed to draw more definitive conclusions, such results are intriguing since suggest a potential role of two genes implicated in neuroplasticity and inflammatory processes in symptom remission after antidepressant treatment.

Case-control association study of 36 single-nucleotide polymorphisms within 10 candidate genes for major depression and bipolar disorder

In this study we investigated 36 single nucleotide polymorphisms within 10 genes previously associated with major depression and bipolar disorder, as well as with the response to their treatment (ABCB1, ABCB4, TAP2, CLOCK, CPLX1, CPLX2, SYN2, NRG1, 5HTR1A and GPRIN2). No association with mood disorders and clinical outcomes was observed.

Influence of COX-2 and OXTR polymorphisms on treatment outcome in treatment resistant depression

Inflammatory pathways play a crucial role in the pathomechanisms of antidepressant efficacy. The aim of this study was to investigate whether a set of single nucleotide polymorphisms (SNPs) within cyclooxygenase-2 (COX-2, rs5275 and rs20417) and oxytocin receptor (OXTR, rs53576 and rs2254298) genes was associated with antidepressant treatment resistance, response or remission. Three hundred seventy-two patients were recruited in the context of a multicenter resistant depression study. They were genotyped for COX-2 and OXTR SNPs. Treatment resistance (according to two different definitions), response and remission were recorded. We did not observe any association between the genotypes or alleles of the selected SNPs within COX-2 and OXTR genes and treatment resistance, response and remission in the whole sample. Our results are consistent with those of some studies but not with those of other ones. Indeed, several factors could be involved in the discrepancy observed across studies. They include sample size, environmental factors, differences in ethnicity, different study designs, and different definitions of treatment resistance.

FMO3 allelic variants in Sicilian and Sardinian populations: trimethylaminuria and absence of fish-like body odor.

The N-oxygenation of amines by the human flavin-containing monooxygenase (form 3) (FMO3) represents an important means for the conversion of lipophilic nucleophilic heteroatom-containing compounds into more polar and readily excreted products. In healthy individuals, virtually all Trimethylamine (TMA) are metabolized to Trimethylamine N-oxide (TMAO). Several single nucleotide polymorphisms (SNPs) of the FMO3 gene have been described and result in an enzyme with decreased or abolished functional activity for TMA N-oxygenation thus leading to TMAU, or fish-like odor syndrome. Three coding region variants, c. G472A (p.E158K) in exon 4, c. G769A (p.V257M) in exon 6, and c.A923G (p.E308G) in exon 7, are common polymorphisms identified in all population examined so far and are associated with normal or slightly reduced TMA N-oxygenation activity. However, simultaneous occurrence of 158K and 308G variants results in a more pronounced decrease in FMO3 activity. A fourth polymorphism, c. G1424A (p.G475D) in exon 9, less common in the general population, was observed in individuals suffering severe or moderate trimethylaminuria. The aim of this study was to determine the allelic and genotypic distributions of these four FMO3 variants in 528 healthy individuals collected from the Sicilian and Sardinian populations together with haplotype and linkage analyses. Finally, we present data on the genotype-phenotype correlation by ESI-MS/MS TMA/TMAO urinary determination in 158KK/308EG individuals. Variant 158K shows the same frequency in Sicilian and Sardinian populations while variant 257M was not observed in the Sardinian sampling. No significant differences were found for 308G and 475D variants among two populations. Cis-linkage between 158K and 308G was confirmed with the compound variant (158K-308G) being found in a proportion of 0.9% and 0.3% of Sicilian subjects, and 0.01% and 0.5% in Sardinian population. Urinary determination of TMA/TMAO ratio in 158KK/308EG individuals showed a considerable reduction in FMO3 activity although they do not show the classical features of trimethylaminuria as a strong body odor and breath. Our data support the conclusion that trimethylaminuria is not always accompanied by a fish-like odor, despite the coexistence in the same individual of the two variants 158K and 308G, and other factors account for the expression of that phenotype.

CCM2 gene polymorphisms in Italian sporadic patients with cerebral cavernous malformation: A case-control study

Cerebral cavernous malformations (CCMs) are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities that can occur sporadically or as a familial autosomal dominant condition with incomplete penetrance and variable clinical expression attributable to mutations in three different genes: CCM1 (Krit1), CCM2 (MGC4607) and CCM3 (PDCD10). Among our group of CCM Italian patients, we selected a cohort of sporadic cases negative for mutations in CCM genes. In this cohort, five variants in CCM2 gene were detected, which proved to be the known polymorphisms in intronic regions (IVS2-36A>G and IVS8 +119 C>T) and in coding sequence (c.157 G>A in exon 2, c.358 G>A in exon 4 and c.915 G>A in exon 8). Therefore, we undertook a case-control study to investigate the possible association of these polymorphisms with sporadic CCMs. The five polymorphisms were identified in 91 CCM sporadic patients and in 100 healthy controls by direct sequencing methods using lymphocyte DNA. Polymorphisms IVS2-36A>G and c.915 G>A showed statistically significant differences in frequencies between patients and controls [(χ2, 6.583; P<0.037); (χ2, 14.205; P<0.001)]. The prevalence of the wild-type genotype was significantly lower in the CCM group than in the control sample. Patients with the A/G and G/G genotypes (IVS2-36A>G) had a significant increase for CCM risk (OR, 3.08; 95% CI, 1.5-5.9 and OR, 4.3; 95% CI, 1.4-22.6) and the same was observed for the polymorphism c.915 G> A (genotype G/A OR, 6.1; 95% CI, 3.0-12.6 and genotype A/A OR, 2.79). In addition, the polymorphisms c.358 G>A in exon 4 (χ2, 15.977; P<0.04) and c.915 G>A in exon 8 (χ2, 18.109; P<0.02) were significantly associated with different types of symptoms. Haplotype analysis, performed only on polymorphisms c.358 G>A (p.Val120Ile), c.915 G>A (p.Thr305 Thr) and IVS2-36A>G, shows that haplotype GAG (+--) significantly increased among CCM sporadic patients compared to the control group. Significant differences between patients and controls were observed only for IVS2-36A>G and c.915 G>A polymorphisms indicating their possible association with sporadic CCMs and an increased risk of CCM. On the other hand, polymorphisms c.358 G>A and c.915 G>A were associated with a more benign course of the disease. These data were confirmed by the haplotype GAG (+--) frequencies.