MaiBritt Giacobini

Localization of cellular retinoid-binding proteins suggests specific roles for retinoids in the adult central nervous system

Retinoic acid, the active metabolite of retinoids (vitamin A compounds), is thought to act as a gene regulator via ligand-activated transcription factors. In order to investigate possible roles of retinoids and retinoid-controlled gene expression in brain function, we have used immunohistochemistry to localize the possible presence of two intracellular retinoid-binding proteins, cellular retinol-binding protein type I and cellular retinoic acid-binding protein type I, in the adult rat central nervous system. We find a widespread, yet distinct, presence of these two binding proteins in the brain and spinal cord. Most of the immunoreactivity is neuronal, including cell somata, as well as dendritic and axonal processes and axon terminals. Cellular retinol-binding protein type I-immunoreactivity is also found in the walls of cerebral blood vessels, the meninges, the choroid plexus, certain ependymal cells, tanocytes and certain other glial elements. The cellular retinol-binding protein type I- and cellular retinoic acid-binding protein type I-immunoreactivity patterns appear to be almost exclusively non-overlapping. Very strong cellular retinol-binding protein type I-immunoreactivity is found in the dendritic layers of the hippocampal formation and dentate gyrus. Cellular retinol-binding protein type I-immunoreactivity is also present in layer 5 cortical pyramidal neurons and neurons in the glomerular layer of the olfactory bulb. Many other areas, e.g. hypothalamic nuclei and amygdala areas, contain networks of varicose cellular retinol-binding protein type I-immunoreactive nerve fibers. The medial amygdaloid nucleus contains strongly cellular retinol-binding protein type I-positive neurons. Cellular retinoic acid-binding protein type I-immunoreactivity is more restricted in the adult brain. Strong cellular retinoic acid-binding protein type I-immunoreactivity is, however, found in a population of medium-sized neurons scattered throughout the striatum, in neurons in the glomerular layer of the olfactory bulb, the olfactory nerve and in a group of nerve cells close to the third ventricle in hypothalamus. The remarkably selective patterns of cellular retinol-binding protein type I- and cellular retinoic acid-binding protein type I-immunoreactivity discovered in the adult rat brain suggest that retinoids have important roles as regulators of gene expression in normal brain function. The high levels of cellular retinol-binding protein type I-immunoreactivity found in hippocampus suggest that one such role might relate to brain plasticity.

Mutation screening of melatonin-related genes in patients with autism spectrum disorders

BackgroundOne consistent finding in autism spectrum disorders (ASD) is a decreased level of the pineal gland hormone melatonin and it has recently been demonstrated that this decrease to a large extent is due to low activity of the acetylserotonin O-methyltransferase (ASMT), the last enzyme in the melatonin synthesis pathway. Moreover, mutations in the ASMT gene have been identified, including a splice site mutation, that were associated with low ASMT activity and melatonin secretion, suggesting that the low ASMT activity observed in autism is, at least partly, due to variation within the ASMT gene.MethodsIn the present study, we have investigated all the genes involved in the melatonin pathway by mutation screening of AA-NAT (arylalkylamine N-acetyltransferase), ASMT, MTNR1A, MTNR1B (melatonin receptor 1A and 1B) and GPR50 (G protein-coupled receptor 50), encoding both synthesis enzymes and the three main receptors of melatonin, in 109 patients with autism spectrum disorders (ASD). A cohort of 188 subjects from the general population was used as a comparison group and was genotyped for the variants identified in the patient sample.ResultsSeveral rare variants were identified in patients with ASD, including the previously reported splice site mutation in ASMT (IVS5+2T>C). Of the variants affecting protein sequence, only the V124I in the MTNR1B gene was absent in our comparison group. However, mutations were found in upstream regulatory regions in three of the genes investigated, ASMT, MTNR1A, and MTNR1B.ConclusionsOur report of another ASD patient carrying the splice site mutation IVS5+2T>C, in ASMT further supports an involvement of this gene in autism. Moreover, our results also suggest that other melatonin related genes might be interesting candidates for further investigation in the search for genes involved in autism spectrum disorders and related neurobehavioral phenotypes. However, further studies of the novel variants identified in this study are warranted to shed light on their potential role in the pathophysiology of these disorders.

Copy Number Variation Characteristics in Subpopulations of Patients With Autism Spectrum Disorders

Autism spectrum disorders (ASDs) are a heterogeneous group of disorders with a complex genetic etiology. We used high‐resolution whole genome array‐based comparative genomic hybridization (array‐CGH) to screen 223 ASD patients for gene dose alterations associated with susceptibility for autism. Clinically significant copy number variations (CNVs) were identified in 18 individuals (8%), of which 9 cases (4%) had de novo aberrations. In addition, 20 individuals (9%) were shown to have CNVs of unclear clinical relevance. Among these, 13 cases carried rare but inherited CNVs that may increase the risk for developing ASDs, while parental samples were unavailable in the remaining seven cases. Classification of all patients into different phenotypic and inheritance pattern groups indicated the presence of different CNV patterns in different patient groups. Clinically relevant CNVs were more common in syndromic cases compared to non‐syndromic cases. Rare inherited CNVs were present in a higher proportion of ASD cases having first‐ or second‐degree relatives with an ASD‐related neuropsychiatric phenotype in comparison with cases without reported heredity (P = 0.0096). We conclude that rare CNVs, encompassing potential candidate regions for ASDs, increase the susceptibility for the development of ASDs and related neuropsychiatric disorders giving us further insight into the complex genetics underlying ASDs.

Role of growth factors in degeneration and regeneration in the central nervous system; clinical experiences with NGF in Parkinson's and Alzheimer's diseases

Neurotrophin-mediated mechanisms are integral to development and maintenance of the adult central nervous system. Neurotrophin expression has been shown to change rapidly in response to many different types of neuronal stress such as excitotoxic injury, mechanical lesions, epileptogenesis and ischemia. It therefore appears as if they are not only to be regarded as target-derived trophic factors in the classical sense, but also as providers of local trophic support and neuronal protection. These discoveries suggest that neurotrophins or compounds with neurotrophin-like actions might become useful in developing new treatment strategies, not only for neurodegenerative diseases, but also for other diseases and injuries to the nervous system including stroke.

Truncated IGF-1 exerts trophic effects on fetal brain tissue grafts

Truncated IGF-1 (tIGF-1), a form of IGF-1 identified in the human brain, has been suggested, from in vitro experiments, to exert neurotrophic effects on developing fetal brain tissue. We studied the effects of tIGF-1 and IGF-1 on small defined areas of the developing central nervous system by using the in vivo model of intraocular transplantation which allows for direct observations of graft survival and growth. Truncated IGF-1 was found to significantly enhance the growth of fetal spinal cord (Embryonic Day (E) 14) and parietal cortex (E16-17) grafts transplanted to the anterior chamber of the eye of adult rats. tIGF-1 increased the volume of cerebral cortex grafts by approximately 100% and of E14 spinal cord grafts by approximately 50%. E18 spinal cord grafts and hippocampal grafts were not stimulated by tIGF-1 as compared to controls given HSA. Effects in cortex were seen with tIGF-1 using concentrations down to at least 10 ng/microliters. Interestingly, intact IGF-1 had no effect on cortical grafts. These findings show for the first time, using an in vivo system, that tIGF-1 is a potent stimulator of growth of grafted fetal cortex cerebri and spinal cord and suggest a possible role for endogenous tIGF-1 in cortical and spinal cord development.

Acidic and basic fibroblast growth factors augment growth of fetal brain tissue grafts

SummaryThe fibroblast growth factor family of peptides (FGFs) are biological regulators which have a diverse array of activities. Among the biological responses reported are inductive effects during early embryogenesis, mitogenic activity on a variety of mesenchymally derived tissues, potent angiogenic activity and neurotrophic activity for both the peripheral and central nervous system. In vitro studies have been performed showing that the FGFs play a regulatory role in the survival and growth of neurons from several regions of the developing rat brain. By using the in vivo model of intraocular transplantation and repeated injections into the anterior chamber, we have been able to observe and follow the survival and growth of small, defined areas of central nervous system (CNS) under the influence of acidic (a) FGF or basic (b) FGF. Acidic FGF significantly enhanced growth of transplanted parietal cortex, embryonic day 17–20 [E17–20], hippocampus [E20] but not spinal cord [E14] when compared to the bovine serum albumin (BSA) vehicle alone. Parietal cortex grafts increased approximately 200% and the hippocampus grafts 100% when stimulated with aFGF. Basic FGF greatly enhanced the growth of intraocularly transplanted parietal cortex (E17–18), hippocampus (E16–17), and spinal cord (E14) by approximately 400%, 100% and 50% respectively when compared to the vehicle alone, and was thus significantly more potent than aFGF at the same concentration. Effects on all areas were seen using concentrations of aFGF down to 25 μg/ml and bFGF as low as 2.5 μg/ml. Histochemical and immunohistochemical studies carried out on cryostat sectioned grafts suggested either no change or normalization of markers for vascularization, glial and neuronal populations. On the basis of these data, we conclude that both aFGF and bFGF may have neurotrophic effects on several brain areas during development.

Differential effects of platelet-derived growth factor isoforms on dopamine neurons in vivo: -BB supports cell survival, -AA enhances fiber formation

Trophic effects of platelet-derived growth factor -AA and -BB on developing (embryonic day 14) ventral mesencephalon were studied using the in vivo model of intraocular transplantation to sympathetically denervated host eyes. This model enabled studies of survival and growth of grafted brain tissue, dopaminergic fiber outgrowth from the grafts onto the host iris as well as morphological effects of platelet-derived growth factor on grafted tissue using markers for tyrosine hydroxylase and glial fibrillary acid protein. Growth of grafts was followed by repeated observations directly through the cornea of the host using a stereomicroscope. This revealed that there was no apparent effect on volume increase of mesencephalic grafts after treatments with either platelet-derived growth factor-AA (100 ng/ml buffer), platelet-derived growth factor-BB (100 ng/ml buffer) or vehicle solution (high salt buffer) alone. Growth factor treatments were administered immediately prior to grafting by incubating the grafts in the appropriate factor as well as on days 5, 10 and 15 postgrafting by administration of 5-microliters intraocular injections of similar solutions as used for incubation. Platelet-derived growth factor-AA significantly enhanced dopaminergic fiber outgrowth from mesencephalic grafts when compared to both platelet-derived growth factor-BB and controls, without an accompanying rise in the number of tyrosine hydroxylase-positive neurons. In contrast, a significantly greater number of tyrosine hydroxylase-positive neurons was seen in grafts treated with platelet-derived growth factor-BB but without an accompanying increase in outgrowth of dopamine-containing fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Chapter 7 Grafts, growth factors and grafts that make growth factors

Publisher Summary This chapter presents an in vivo screening system based upon the intraocular grafting of fetal brain tissue, which can be used to monitor temporal and regional specificity of growth factor effects. It focuses on the most well-known nerve growth stimulating factor, nerve growth factor (NGF), and describes its presence in the central nervous system (CNS) as well as the fate of intraparenchymally injected NGF. The fate of NGF injected directly into brain parenchyma as monitored by immunohistochemical techniques is described in the chapter. Genetically engineered fibroblasts secreting large amounts of NGF that have been grafted to the CNS explain the way such cells can support surrounding cholinergic systems and rescue axotomized cholinergic neurons. The functions of NGF in the CNS might extend beyond the current set of cholinergic neurons. Thus, it is conceivable that NGF sensitivity is more widespread during development.

Association of adenomatous polyposis coli (APC) gene polymorphisms with autism spectrum disorder (ASD).

We serendipitously identified a single nucleotide polymorphism (SNP), 8636C>A (rs1804197) in the 3′‐untranslated region of the adenomatous polyposis coli (APC) gene to be associated with autism spectrum disorder (ASD). In order to gain further evidence for the association between the APC locus and ASD, we genotyped four additional adjacent common SNPs (rs2229992, rs42427, rs459552, and rs465899) in the coding regions within the APC gene in a set of Swedish ASDs and controls. One common haplotype TGAG was found to be associated with ASD after haplotype analysis using both Haploview v3.1.1 (P = 0.006) and COCAPHASE v2.403 (P = 0.030). This result is the first to suggest that the genomic locus at APC is associated with ASD, and that the APC gene itself is a good predisposing candidate to be evaluated in future studies due to its important role in neuronal development and function.

Differential effects of platelet-derived growth factors on fetal hippocampal and cortical grafts : evidence from intraocular transplantation in rats

Effects of platelet-derived growth factor-AA (PDGF-AA) and platelet-derived growth factor-BB (PDGF-BB) on developing parietal cortex (E16) and hippocampal (E18-E19) grafts were studied using the in vivo method of intraocular transplantation. Survival and growth of grafts in the anterior eye chamber of adult host rats under the influence of regular treatments with 0.5 ng (in a 100 ng/ml concentration) PDGF-AA or PDGF-BB was followed and compared to those receiving vehicle solution alone (0.5 mg HSA/ml Hanks). Both PDGF-AA and PDGF-BB increased the volume of transplanted cortical grafts. PDGF-BB also exerted trophic effects on grafted hippocampal tissue whereas PDGF-AA seemed to inhibit hippocampal growth. Histological and immunohistochemical studies revealed an increase in the density of astroglial elements in PDGF-AA- and PDGF-BB-treated cortical grafts whereas the PDGF-AA- and PDGF-BB-treated hippocampal grafts maintained a cytoarchitecture closely resembling that of control grafts. These findings support in vitro experiments showing that developing glial cells are stimulated by PDGFs and we further propose regional differences of action of PDGFs in the developing central nervous system.