Plasticity of perivascular nerves

This thesis presents a study on the plasticity of perivascular nerves, and is divided in two sections. The first concerns changes in expression of perivascular nerves during development and ageing and in the nerves that remain after selective sympathetic and/or sensory denervation of the rat cerebral, meningeal and irideal vessels. In the second section changes in the innervation of the guinea pig uterine artery during pregnancy and after chronic treatment with progesterone are described. Changes in the expression of neurotransmitters in cerebrovascular nerves were studied in rats from birth to 27 months of age, using histochemical and immunohistochemical techniques, followed by quantitative analysis of the density of innervation. The study revealed an early development of sympathetic cerebrovascular nerves, while sensory nerves and parasympathetic nerves developed later. Ageing resulted in a decrease of the expression of vasoconstrictor neurotransmitters in cerebrovascular nerves, whereas the expression of vasodilator neurotransmitters (vasoactive intestinal polypeptide, VIP, and calcitonin gene-related peptide, CGRP) was strikingly increased. Long-term sympathectomy produced by chronic treatment with guanethidine caused degeneration of the sympathetic neurons in the superior and inferior cervical ganglia and disappearance of glyoxylic acid-induced fluorescent nerves and 5-hydroxydopamine (5-OHDA) labelled vesicles in rat cerebral, meningeal and irideal vessels. The results also suggest that there is a compensatory increase of the expression of neuropeptide Y (NPY)-immunoreactivity (IR) in non-sympathetic perivascular axons in the brain and iris of developing rats subjected long-term guanethidine treatment, but not in those of the dura mater. Using a dual immunostaining technique at the ultrastructural level, the NPY-IR present in cerebrovascular nerves of developing rats sympathectomized with long-term guanethidine treatment was localized in VIP-IR nerve fibres. Their origin, from pterygopalatine ganglion neurons, was traced using a fluorescent neuronal tracer (fast blue) and immunohistochemistry revealed an increase in the expression of NPY-IR in pterygopalatine ganglion neurons after long-term sympathectomy. An increased number of CGRP- and substance P (SP)-IR nerves was observed in the anterior cerebral artery, iris and dura mater of guanethidine sympathectomized rats. These nerve fibres could arise from sensory and/or parasympathetic cranial neurons storing CGRP- and/or SP-LI. Rats were also subjected to long-term guanethidine treatment afer capsaicin treatment. Projection of sensory CGRP- , but not SP-IR neurons which did not degenerate after capsaicin treatment again increased dramatically after guanethidine treatment. In the second section of this thesis, the number of noradrenaline (NA)-containing nerves in the uterine artery, which formed the densest plexus in virgin animals, was much reduced in late pregnancy, a finding supported by a significant reduction in noradrenaline levels. In contrast, the innervation of the uterine artery by NPY-IR nerve fibres was increased in pregnancy, while the other peptidergic nerves and peptide levels were unchanged. Systemic progesterone treatment did not mimic these changes. An immunocytochemical analysis of uterine arteries from virgin and pregnant guinea-pigs injected with 5-OHDA to label noradrenergic nerves showed that, unlike the pregnant uterus, no degeneration of nerves containing large and small dense-cored vesicles occurred in pregnant uterine arteries. The results suggested that, in pregnant animals, 5-OHDA labelled vesicles were also present in non-noradrenergic perivascular nerves. In the uterine arteries from pregnant guinea-pigs, the neuronal uptake of 3H-NA was found to be increased compared with that of control arteries, despite the reduction of noradrenergic nerves. The autoradiographic localization of 3H-NA taken up by guinea pig uterine arteries, further processed for immunocytochemistry of dopamine β hydroxylase (DBH)-, NPY-, VIP-, SP- and CGRP-IR, revealed that the uptake of 3H-NA in pregnant animals occurred also in non-sympathetic nerves. This study showed that : a) compensatory changes in the expression of neurotransmitters in perivascular nerves occur both in physiological (development, aging, pregnancy) and experimental (selective denervation) conditions; b) the changes that have been observed are complex: one clear example is a marked increase in CGRP/SP in sensory nerves following destruction of sympathetic nerves; another is the increase of NPY in perivascular nerves supplying the uterine artery in late pregnancy; c) plasticity is a property of perivascular nerves that is retained throughout life

The zebrafish as an emerging model to study DNA damage in aging, cancer and other diseases

Cancer is a disease of the elderly, and old age is its largest risk factor. With age, DNA damage accumulates continuously, increasing the chance of malignant transformation. The zebrafish has emerged as an important vertebrate model to study these processes. Key mechanisms such as DNA damage responses and cellular senescence can be studied in zebrafish throughout its life course. In addition, the zebrafish is becoming an important resource to study telomere biology in aging, regeneration and cancer. Here we review some of the tools and resources that zebrafish researchers have developed and discuss their potential use in the study of DNA damage, cancer and aging related diseases

Rad21 Haploinsufficiency Prevents ALT-Associated Phenotypes in Zebrafish Brain Tumors

Cohesin is a protein complex consisting of four core subunits responsible for sister chromatid cohesion in mitosis and meiosis, and for 3D genome organization and gene expression through the establishment of long distance interactions regulating transcriptional activity in the interphase. Both roles are important for telomere integrity, but the role of cohesin in telomere maintenance mechanisms in highly replicating cancer cells in vivo is poorly studied. Here we used a zebrafish model of brain tumor, which uses alternative lengthening of telomeres (ALT) as primary telomere maintenance mechanism to test whether haploinsufficiency for Rad21, a member of the cohesin ring, affects ALT development. We found that a reduction in Rad21 levels prevents ALT-associated phenotypes in zebrafish brain tumors and triggers an increase in tert expression. Despite the rescue of ALT phenotypes, tumor cells in rad21+/− fish exhibit an increase in DNA damage foci, probably due to a reduction in double-strand breaks repair efficiency

A network-based approach to identify deregulated pathways and drug effects in metabolic syndrome

Metabolic syndrome is a pathological condition characterized by obesity, hyperglycemia, hypertension, elevated levels of triglycerides and low levels of high-density lipoprotein cholesterol that increase cardiovascular disease risk and type 2 diabetes. Although numerous predisposing genetic risk factors have been identified, the biological mechanisms underlying this complex phenotype are not fully elucidated. Here we introduce a systems biology approach based on network analysis to investigate deregulated biological processes and subsequently identify drug repurposing candidates. A proximity score describing the interaction between drugs and pathways is defined by combining topological and functional similarities. The results of this computational framework highlight a prominent role of the immune system in metabolic syndrome and suggest a potential use of the BTK inhibitor ibrutinib as a novel pharmacological treatment. An experimental validation using a high fat diet-induced obesity model in zebrafish larvae shows the effectiveness of ibrutinib in lowering the inflammatory load due to macrophage accumulation

Lactate modulates microglia polarization via IGFBP6 expression and remodels tumor microenvironment in glioblastoma

Lactic acidosis has been reported in solid tumor microenvironment (TME) including glioblastoma (GBM). In TME, several signaling molecules, growth factors and metabolites have been identified to induce resistance to chemotherapy and to sustain immune escape. In the early phases of the disease, microglia infiltrates TME, contributing to tumorigenesis rather than counteracting its growth. Insulin-like Growth Factor Binding Protein 6 (IGFBP6) is expressed during tumor development, and it is involved in migration, immune-escape and inflammation, thus providing an attractive target for GBM therapy. Here, we aimed at investigating the crosstalk between lactate metabolism and IGFBP6 in TME and GBM progression. Our results show that microglia exposed to lactate or IGFBP6 significantly increased the Monocarboxylate transporter 1 (MCT1) expression together with genes involved in mitochondrial metabolism. We, also, observed an increase in the M2 markers and a reduction of inducible nitric oxide synthase (iNOS) levels, suggesting a role of lactate/IGFBP6 metabolism in immune-escape activation. GBM cells exposed to lactate also showed increased levels of IGFBP6 and vice-versa. Such a phenomenon was coupled with a IGFBP6-mediated sonic hedgehog (SHH) ignaling increase. We, finally, tested our hypothesis in a GBM zebrafish animal model, where we observed an increase in microglia cells and igfbp6 gene expression after lactate exposure. Our results were confirmed by the analysis of human transcriptomes datasets and immunohistochemical assay from human GBM biopsies, suggesting the existence of a lactate/IGFBP6 crosstalk in microglial cells, so that IGFBP6 expression is regulated by lactate production in GBM cells and in turn modulates microglia polarization

Fam83F induces p53 stabilisation and promotes its activity

p53 is one of the most important tumour suppressor proteins currently known. It is activated in response to DNA damage and this activation leads to proliferation arrest and cell death. The abundance and activity of p53 are tightly controlled and reductions in p53's activity can contribute to the development of cancer. Here, we show that Fam83F increases p53 protein levels by protein stabilisation. Fam83F interacts with p53 and decreases its ubiquitination and degradation. Fam83F is induced in response to DNA damage and its overexpression also increases p53 activity in cell culture experiments and in zebrafish embryos. Downregulation of Fam83F decreases transcription of p53 target genes in response to DNA damage and increases cell proliferation, identifying Fam83F as an important regulator of the DNA damage response. Overexpression of Fam83F also enhances migration of cells harbouring mutant p53 demonstrating that it can also activate mutant forms of p53