Athina Samara

Molecular understanding of lung development is crucial for developing therapies and diagnostic tools. Animal models with altered thyroid hormone signaling provide mechanistic insight into thyroid-dependent neonatal lung disease. Repression of Klf2 (Krüppel-like factor 2), a suggested T3 target gene, is associated with disrupted lung development in mice. Klf2 is proposed to be specifically involved in type I pneumocyte differentiation. To explore mechanisms of thyroid-dependent lung disease, we studied developing chicken fetuses with experimentally induced hypothyroidism. Morphology and the expression of a panel of molecules linked to Klf2 were assessed using histology, immunohistochemistry, Western blot and qPCR. Methimazole injections at E14 hampered lung maturation. The effects of methimazole were evident in several tissue compartments, and impacted on both pneumocyte and vascular differentiation, suggesting cellular and molecular pleiotropy. Concomitant expression changes in a pa...

CHARACTERIZATION OF DOPA-ERGIC PROPERTIES OF IMMORTALIZED NEUROENDOCRINE NEURONS Samara A, Bondiolotti GP*, Ranauro M, Giacobini P°, Maggi R Lab. Dev. Neuroendocrinology, Dept. of Endocrinology, Centre of Excellence on Neurodegenerative Diseases, *Dept. of Pharmacology, ; University of Milano, Italy; °Dept. of Human and Animal Biology, University of Turin, Torino, Italy 3,4-dihydroxyphenylalanine (DOPA) has been believed to be an inert amino acid, synthesized from tyrosine by the action of the tyrosine hydroxylase (TH), that alleviates the symptoms of Parkinson's disease by its conversion to dopamine via the enzyme aromatic L-amino acid decarboxylase (AADC). Nevertheless, it has been proposed an active role of DOPA as neurotransmitter/neuromodulator (Misu et al., Pharm Ther, 2003); actually, DOPA has been found to modulate dopamine and noradrenaline release, in presence of AADC inhibitors, by interaction with presynaptic D2 dopamine receptors. Moreover, DOPA-ergic neurons have b...

MTA, Bio-Oss, and dentin chips have been successfully used in endodontics. The aim of this study was to assess the adhesion and migration of dental stem cells on human pulp ceiling cavities filled with these endodontic materials in an experimental model, which mimics the clinical conditions of regenerative endodontics. Cavities were formed, by a homemade mold, on untouched third molars, filled with endodontic materials, and observed with electron microscopy. Cells were seeded on cavities’ surface and their morphology and number were analysed. The phenomenon of tropism was assessed in a migration assay. All three materials demonstrated appropriate microstructures for cell attachment. Cells grew on all reagents, but they showed a differential morphology. Moreover, variations were observed when comparing cells numbers on cavity’s filling versus the surrounding dentine disc. The highest number of cells was recorded on dentin chips whereas the opposite was true for Bio-Oss. This was conf...

Seladin-1/DHCR24 is the enzyme that reduces the 24-25 double bond of all Delta-24 sterols, and converts desmosterol to cholesterol. Alterations in Seladin-1 expression have been related to Alzheimer’s disease, aging, cellular senescence, apoptosis, resistance or vulnerability to oxidative stress, fertility, dermopathies and oncogenesis. Piling evidence suggests DHCR24 is involved in various physiological processes and pathological conditions, through actions that transcend its enzymatic role in cholesterol biosynthesis. Neutralizing mutations of the DHCR24 gene cause desmosterolosis, a rare recessive autosomal potentially lethal disorder. Only two patients with desmosterolosis have been reported, sharing clinical features with other post-squalenic pathway disorders. These manifestations included macrocephaly, cleft palate, nasal hypoplasia, short limbs but no polydactyly or syndactyly and generalized osteosclerosis, and agenesis of the corpus callosum. In the present work Zebrafish ...

The recently cloned DHCR24 is the enzyme that converts desmosterol to cholesterol and has been associated with neuroprotection, neurodegeneration, and tumourigenesis. Inactivating mutations of the gene cause desmosterolosis. Only two patients have been reported and the craniofacial and neurodevelopmental anomalies of desmosterolosis in them reveal the importance of cholesterol in prenatal development. Interestingly, DHCR24 KO mice surpassed embryonic stages & died perinatally. Blocking DHCR24 gene translation by morpholinos at the one-cell stage of zebrafish eggs, showed that DHCR24 affects brain development. A more thorough study of the morphants revealed neural defects and muscle disorganization, pigmentation defects and pharyngeal arch deformities. Overall the data unveiled an essential role for DHCR24 in early morphogenesis. Although data from human patients are limited, results with zebrafish knockdowns matched the phenotype and demonstrated other defects such as microcephaly, ...

It has been recently demonstrated that stem cells exist in all tissues of the human organism, including the dental pulp. Four different types of dental stem cells have been isolated from dental tissue. According to their origin, they are divided in: (i) Dental pulp stem cells (DPSCs), (ii) Stem cells from human exfoliated teeth (SHED), (iii) Stem cells from apical papillae (SCAP) and (iv) periodontal ligament stem cells (PDLSCs). The various dental stem cell populations have a differential multilineage differentiation potential with variable efficiency, whereas diversity is also observed in the protein expression and in the gene profile. Even though a detailed characterization of these different subpopulations has been attempted the last decades, the results of the studies are not always clear and consistent. Therefore, further characterization is required and more importantly, their response in the presence of various dental materials remain unraveled. We aim to address the above t...

X-linked Kallmann's syndrome (KS) is a genetic disease characterized by anosmia and hypogonadism due to the impairment of olfactory axons development and of LHRH neurons migration. A gene located at Xp22.3 (KAL-1) appears to be responsible for the X-linked KS; it encodes for a secreted heparin binding-protein (KAL-1 or anosmin), which exhibits similarities with cell-adhesion molecules. KAL was found to play a role in the targeting of olfactory axons; however, no data are so far available on its effect on the migration of LHRH neurons. In order to clarify this issue, we exposed GN11 cells (a cell line of immortalized migrating LHRH neurons), in a microchemotaxis chamber, to conditioned media (CM) of COS cells transfected with a myc-tagged hKAL-1 expression vector (COSKAL) or with three forms of KAL-1 carrying different missense mutations (N267K, E514K, F517L) found in patients affected by KS. We found that the presence of KAL protein in the CM significantly increases the chemotac...

Seladin-1 stands for Selective Alzheimer’s Disease Indicator-1. By sequence similarity, it was identified as DHCR24; the enzyme that reduces the C24-C25 double bond of desmosterol or other delta-24 sterol intermediates producing cholesterol. It is highly expressed in the brain, conserved from plants to mammals. Its inactivating mutations are the genetic basis of desmosterolosis, disease characterized by desmosterol accumulation and neurological defects. Relative to Alzheimer’s disease (AD), Seladin-1 mRNA is down-regulated in the brain regions more susceptible to the AD pathology. Seladin-1 has been implicated with neuroprotection and oxidative stress, suggesting a role in cell survival. Seladin-1 expression in isolated primary neurons (PN) had not been investigated. In addition, with the cholesterol supply to neurons being mediated by astrocytes, the latter were also considered a key element to study. Experiments carried out, both in rat and mouse, evaluated the expression of Selad...

One of the commonly used animal models in fertility, developmental and neurobiological studies is the laboratory rat. The early recognition of rat pregnancy and confirmation of the exact embryonic day are vital. The aim of this study was to investigate the correlation of maternal weight at the time of conception to its increase throughout gestation, aiming to develop a mathematical model, which can be used for the determination of the exact day of pregnancy, set the threshold, and monitor pregnancy from the onset. We studied a total of 173 Wistar rats with a mean body weight of 238.22 ± 34.9 g. After 72 h at the male's cages, we considered as Day 0 (D0) the day in which a copulatory plug or sperm was found during the vaginal smear examination. After that period the female animals were transferred into their cages, and weight monitoring started 14 days (D14) after D0, until parturition. Based on the statistical analysis, there is a correlation between maternal body weight at D0 a...

Promoter regions of the human genome play a key role in our understanding of the regulatory mechanisms related to the physiological and disease states. The aim of this study was to investigate the sequence positional properties of experimentally verified human promoters. Consequently, we determined short sequence elements ranging from 4 to 9mers presenting position dominance close to, or away from the transcription start site (TSS). For this purpose rigid statistical criteria were used and whether position dominance was in any way related to transcription control was determined. To achieve this goal we designed and implemented a dedicated filtering method to massively detect position-dominant sequence elements embedded in the promoter set. Additionally, via a high throughput procedure, we gathered data on the majority of the publicly available transcription factor-binding sites (TFBSs) and matched them to our findings, aiming to accomplish a large-scale correlation between position-...

Neuroendocrine control of physiological functions needs a complex developmental organisation of the hypothalamic parvicellular neurons, which synthesise and release hypophysiotropic hormones. Among the hypothalamic neuroendocrine cells, Gonadotropin-releasing hormone (GnRH) neurons represent a unique class; they are generated in the olfactory placode and, during embryonic life, migrate to the septo/hypothalamic region along terminal and vomeronasal nerves. At this level GnRH neurons undergo terminal differentiation and start to release GnRH to modulate the secretion of pituitary gonadotropins. All these steps are under the strict control of several developmental cues and their defect might represent a cause of clinical disorders. A number of factors have been proposed to be involved in the migration of GnRH neurons, but their role is still unclear. By using gene knockout techniques it has been found that mice carrying a targeted deletion of Ebf2 gene, a component of Olf/Ebf bHLH tra...

Researchers sometimes face difficulties in the diagnosis of pregnancy and assessment of embryonic development. Ultrasonography (US) is a non-invasive imaging method with minimal side effects on the subjects or operators. It provides real-time evaluation of the physiology of rapidly moving structures (i.e., heart) and facilitates evaluation of fetal tissue development. US discerns tissues based on composition, making it the imaging method of choice for abdominal examination. In this study we used real-time US as an alternative method for early diagnosis of pregnancy in rats. Sixty-four Wistar rats aged 16-20 wk were examined, and day 8 was the earliest point at which pregnancy could be detected. We constructed a detailed timeline of embryonic features detectable by US on days 8 to 19. We trust this index will be a valuable tool. More refined work toward a more detailed "atlas" will help to reduce animal sacrifice during embryonic development studies.

3-betahydroxysterol delta-24-reductase (DHCR24), also called selective Alzheimer's disease indicator-1, is a crucial enzyme in cholesterol biosynthesis with neuroprotective properties that is downregulated in brain areas affected by Alzheimer's disease. In the present study, we investigated modifications of DHCR24 expression in models of Huntington's disease (HD), a neurodegenerative disorder caused by a polyglutamine expansion in huntingtin (Htt) protein that induces degeneration of cerebral cortex and striatum as well as lateral hypothalamic abnormality. Basal expression of DHCR24 and its modulation after oxidative stress were evaluated in rat striatal precursors cells (ST14A) transfected with wild-type (Htt) or mutant Htt (mHtt) and in brain tissue of an HD mouse model (R6/2). The results showed that DHCR24 transcript levels were decreased in ST14A cells expressing mHtt and in the brain of symptomatic R6/2 mice, but were significantly increased in ST14A cells overexpressing wild-type Htt. In addition, we demonstrated that, in the striatal precursors, the decrease of DHCR24 expression in response to oxidative stress was modified according to the presence of Htt or of its mutant form. Preliminary results indicated a modification of DHCR24 expression in post-mortem brain samples of HD patients. In conclusion, these results support the hypothesis of a possible role of DHCR24 in HD.

Primary neurons were grown on structured silicon (Si) substrates, in the absence of chemotropic factors or synthetic extracellular matrix. The Si substrates used for the study comprise hierarchical structures in the micro- and nanolength scales. The substrates were structured via femtosecond laser irradiation of the Si wafer, in a reactive SF(6) environment. Electron microscopy revealed that the neurons formed an elaborate web of cytoplasmic processes in the absence of glial elements. The neuronal cytoplasm autografted the depth of the spikes, and the neurite sprouting took place over the spikes surface. Here we demonstrate how microfabrication of a Si surface provides an excellent platform for multifaceted studies of neuronal specimens.

Prenatal exposure of rodents to glucocorticoids (Gc) affects the sexual development of the offspring, possibly interfering with the differentiation of the hypothalamic-pituitary-gonadal axis. Glucocorticoid receptors (GR) are present on gonadotropin-releasing hormone (GnRH) neurons in the rat hypothalamus, suggesting a direct effect of Gc in the control of the synthesis and/or release of the hormone. In this study, we demonstrate the colocalization of immunoreactive GR with GnRH in a subpopulation of mouse hypothalamic GnRH neurons, confirming the possible involvement of Gc in mouse GnRH neuronal physiology. Receptor-binding assay, RT-PCR, immunocytochemistry, and immunoblotting experiments carried out in GN11 immortalized GnRH neurons show the presence of GR even in the more immature mouse GnRH neurons and confirm the expression of GR in GT1-7 mature GnRH cells. In GN11 cells, the activation of GR with dexamethasone produces nuclear translocation, but does not lead to the inhibition of GnRH gene expression already reported in GT1-7 cells. Long-term exposure of GN11 cells to dexamethasone induces an epithelial-like phenotype with a reorganization of F-actin in stress fibers. Finally, we found that Gc treatment significantly decreases the migratory activity in vitro and the levels of phosphorylated focal adhesion kinase of GN11 immature neurons. In conclusion, these data indicate that GR are expressed in mouse hypothalamic GnRH neurons in vivo as well as in the immature GN11 GnRH neurons in vitro. Moreover, the effects of the GR activation in GN11 and in GT1-7 cells may be related to the neuronal maturational stage of the two cell lines, suggesting a differential role of Gc in neuronal development.

Biological systems demonstrate asymmetry, while lateralization has been observed from humans to lower animals structurally, functionally and behaviorally. This may be derived from evolutionary, genetic, developmental, epigenetic and pathologic factors. However, brain structure and function is complex, and macroscopic or microscopic asymmetries are hard to discern from random fluctuations. In this article, we discuss brain laterality and lateralization, beginning with a brief review of the literature on brain structural and functional asymmetries. We conclude with methods to detect and quantify asymmetry, focusing on neuroproteomics, for retrieval of protein-expression patterns, as a method of diagnosis and treatment monitoring. We suggest inter-hemispheric differential proteomics as a valid method to assess the experimental and biological variations in the healthy brain, and neurologic and neuropsychiatric disorders.

The objective of this study was to investigate the effects of two sources of electromagnetic fields (EMFs) on the proteome of cerebellum, hippocampus, and frontal lobe in Balb/c mice following long-term whole body irradiation. Three equally divided groups of animals (6 animals/group) were used; the first group was exposed to a typical mobile phone, at a SAR level range of 0.17-0.37 W/kg for 3 h daily for 8 months, the second group was exposed to a wireless DECT base (Digital Enhanced Cordless Telecommunications/Telephone) at a SAR level range of 0.012-0.028 W/kg for 8 h/day also for 8 months and the third group comprised the sham-exposed animals. Comparative proteomics analysis revealed that long-term irradiation from both EMF sources altered significantly (p < 0.05) the expression of 143 proteins in total (as low as 0.003 fold downregulation up to 114 fold overexpression). Several neural function related proteins (i.e., Glial Fibrillary Acidic Protein (GFAP), Alpha-synuclein, Glia Maturation Factor beta (GMF), and apolipoprotein E (apoE)), heat shock proteins, and cytoskeletal proteins (i.e., Neurofilaments and tropomodulin) are included in this list as well as proteins of the brain metabolism (i.e., Aspartate aminotransferase, Glutamate dehydrogenase) to nearly all brain regions studied. Western blot analysis on selected proteins confirmed the proteomics data. The observed protein expression changes may be related to brain plasticity alterations, indicative of oxidative stress in the nervous system or involved in apoptosis and might potentially explain human health hazards reported so far, such as headaches, sleep disturbance, fatigue, memory deficits, and brain tumor long-term induction under similar exposure conditions.