The aim of this Thesis is to investigate the use of hyaluronic acid as a material for the design of scaffolds aimed at CNS regeneration. The motivation comes from the need of searching for new strategies that allow regeneration in the central nervous system. In degenerative diseases, such as Parkinson's disease, where the progressive loss of neuronal subpopulations occurs, a permissive environment able to support regeneration and connectivity of neurons from the host tissue may be a promising therapy to recover lost functionalities. In this Thesis we have focused on the development of structures able to integrate within the brain, supporting neural cells attachment and survival. We hypothesized that hyaluronic acid provides an enabling environment and appropriate for regeneration due to its biocompatibility and diverses physiological applications. Biocompatible hydrogels based on modified hyaluronic acid were synthesized. Covalently crosslinked hyaluronic acid hydrogels, alone or in combination with acrylic polymers, were synthesized and permitted to develop different porous structures which may serve in different applications as cell supply, cell repopulation or tissue regeneration. Highly porous with interconnected spherical pores, hollow tubes or multichanneled scaffolds were developed. The processes allow for a wide range of shapes for different applications within the scope of central nervous system regeneration. Furthermore, in vitro culture of human cell lines together with biomaterials was performed. A human microvascular endothelial cell line (hCMEC/D3) and a human glioma cell line (U373) were chosen for the studies. Experiments were focused on the interaction between hyaluronan based scaffolds and those cell lines composing the blood-brain-barrier (BBB) in the central nervous system. Biocompatibility, viability and phenotype characteristics were assessed. / Baiget Orts, MA. (2012). HYALURONAN BASED BIOMATERIALS FOR CENTRAL NERVOUS TISSUE REGENERATION [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/14576 / Palancia

Se pretende obtener un material semibiodegradable basado en ácido hialurónico químicamente enlazado a cadenas de polímeros acrílicos. Los hidrogeles de ácido hialurónico presentan en general buenas características para su utilización en regeneración del sistema nervioso central: es biodegradable, es un componente importante del tejido neural, sus propiedades mecánicas son semejantes a las del tejido cerebral, promueve la formación de nuevos capilares (angiogénesis), y limita la inflamación. Con este nuevo material se pretende mejorar el excesivo grado de hinchado en medio fisiológico, su rápida degradación, mejorar la adhesión celular, además la matriz permanente de las cadenas acrílicas pueden actuar como un soporte permanente durante el proceso regenerativo sin que se produzca una pérdida brusca de propiedades mecánicas y estructurales. El trabajo consiste en caracterizar este nuevo material así como los productos intermedios necesarios para su obtención final, comparándolo con las propiedades de un hidrogel de ácido hialurónico sin incorporar cadenas acrílicas. Los estudios celulares se llevaran a cabo in vitro, como fase preliminar para futuros implantes en el cortex cerebral, estudiando la capacidad de diferenciación de precursores neurales y de generación de nuevos capilares con el fenotipo típico de la barrera hematoencefálica, mediante el estudio de cocultivos de precursores neurales y células endoteliales. / Perez Garnes, M. (2015). Structures based on semi-degradable biomaterials for neural regeneration in the central nervous system [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48799 / TESIS

Chronic kidney disease (CKD), a reduction in kidney function, has reached pandemic proportions and imposes a major healthcare burden worldwide. A hallmark of CKD is the accumulation of several chemical compounds, called uremic toxins, which inflict systemic and renal-specific damage. Of the known uremic toxins, kynurenine (Kyn) is known to be particularly vasculotoxic and is implicated in several complications of CKD. Indoleamine 2,3-dioxygenase 1 (IDO), which catalyzes the first step in the metabolism of Tryptophan (Trp), regulates immune response to inflammatory cytokines in tissues. IDO plays a role in apoptosis and damage during acute kidney injury (AKI), a transient decrease in kidney function. During metabolism of Trp, IDO generates Kyn, a uremic solute, and therefore IDO may play a role in the brain and kidney damage due to accumulation of Kyn. The objective of the current study was to investigate the role and regulation of IDO in CKD pathology. Studies were performed to determine whether IDO is protective or pathologic and to find how IDO is regulated in the kidney during CKD. IDO in renopathology was examined using murine models of CKD. CKD was induced via a 0.2% adenine-supplemented diet (AD) model for 21 days. IDO regulation was examined using an Indoxyl Sulfate (IS)-specific solute model. Renal function in the IDO+/+ and IDO-/- AD mice was assessed through weekly measurement of blood urea nitrogen (BUN). H&E and Masson’s trichrome stains were used to assess percentages of glomerulosclerosis (GS) and immune infiltration (II), and combined interstitial fibrosis and tubular atrophy (IFTA) score in IDO+/+ and IDO-/- mice with and without CKD. IDO protein concentration in the kidneys of all mice with and without CKD and IDO+/+ IS mice was determined via immunoblotting. Patients with kidney disease suffer from neuropsychological disorders and neurocognitive decline. The effects of uremic solutes on the CNS was examined using immortalized human umbilical endothelial vein cells (HUVEC-TERT), in vitro. Cell proliferation and viability, in the presence of IS, were measured by BrdU and Alamar blue assays, respectively. In both IDO+/+ and IDO-/-, 21 days of AD results in significant deterioration of renal function. The average IFTA score and percentage of II in IDO-/- mice increased with AD compared to ND (p<0.05, p<0.001). IDO expression was seen sporadically in the glomeruli and walls of major vessels in the kidneys of 4d AD IDO+/+ mice, and in the tubules and vessel walls in the kidneys of 14d AD IDO+/+ mice. In IDO+/+ ND mice, endogenous IDO protein expression was undetectable at a signal intensity of 119.86 ± 268.01, whereas IDO+/+ AD mice showed a 370-fold higher level of IDO protein expression compared to IDO+/+ ND (p<0.001). IDO-/- AD IDO protein expression was 9.5-fold higher than in IDO-/- AD (p<0.05). IDO expression was found to be 58-fold higher in IDO+/+ mice with IS treatment (p<0.05). In the IS mice, non-significant trends toward decrease in cellular proliferation and viability with time were also observed (p=ns). IDO is upregulated at the protein level both in a CKD model and directly by the uremic solute, IS. IDO appears to be protective in the kidney during CKD, given the trend toward increased percentage of GS and II in IDO-/- compared to IDO+/+ mice with CKD, though there is little difference seen in total kidney IFTA. IDO upregulation is linked to increased apoptosis. Blocking uremic solute production would therefore prevent IDO protein upregulation and reduce apoptosis, alleviating renal damage during CKD.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第21861号 / 人博第890号 / 新制||人||213(附属図書館) / 2018||人博||890(吉田南総合図書館) / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 神﨑 素樹, 教授 石原 昭彦, 教授 久代 恵介 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

N-acetylaspartate (NAA) is one of the most abundant molecules in the mammalian central nervous system (CNS). The current paradigm suggests that NAA is synthesized in neurons by the enzyme N-acetyltransferase 8-like (NAT8L) and hydrolyzed into aspartate and acetate by the enzyme aspartoacylase (ASPA) in oligodendrocytes. Although the function of NAA is not well understood, several hypotheses have been proposed since its discovery several decades ago. Among the most cited theory is the concept of acetate delivery to oligodendrocytes via NAA for the synthesis of fatty acids for myelin lipids and myelination. Another concept suggests that NAA functions as a molecular water pump to remove molecular water from oxidative phosphorylation. In contrast, disruption of NAA metabolism has been associated with oxidative stress contributing to neurodegeneration, as seen in Canavan disease, a monogenic disorder associated with loss-of-function mutations in ASPA. Accumulation of NAA in the CNS and peripheral organs is pathognomonic for Canavan disease (CD) and is used clinically to diagnose this rare disease. Symptoms typically occur within months after birth and primarily manifest in the CNS with spongy degeneration of the white matter. Initially, affected patients present with poor feeding, lack of head control, hydrocephalus; later, they miss developmental milestones and develop seizures. Only supportive treatment is available possibly helping patients to survive past the first couple of years. Gene therapy has been considered early on for the treatment of CD. The first trial in humans demonstrated safety but did not result in symptomatic improvement. In addition to gene therapy for the treatment of CD, NAA has gained increasing interest in neurodegenerative and psychiatric disorders, but also in adipose tissue. Here, we are investigating the function of NAA in the context of ASPA deficiency, aka Canavan disease. We found that impaired NAA metabolism caused by ASPA mutations is characterized by a neurometabolic profile that suggests cellular shift from glucose towards fatty acid metabolism for energy production. Although, we found a similar metabolic signature in asymptomatic mice within days after birth, longitudinal comparison suggest that disease progression leads to fatty acid depletion, which is not present in asymptomatic mice, potentially challenging the concept that NAA-derived acetate is essential for lipid synthesis in the myelinating brain. Using rAAV to determine the reversibility of this metabolic phenotype, we found that early treatment prevents loss of myelin, normalizes the neurometabolic phenotype and keeps Canavan mice asymptomatic; in contrast, later treatment only allows for partial normalization of the neurometabolome, despite adequate ASPA gene delivery by rAAV, independent of ubiquitous or astrocyte-restricted hASPA expression. Furthermore, we found that non-enzymatically active hASPA might play a ubiquitous role in glucose uptake regulation in vivo. Importantly, we identified brain regions with metabolic changes that also correspond to the areas with significant histopathologic alterations. Finally, we confirmed the glycolytic changes in a Canavan disease patient cell line using Seahorse metabolic analyzer, demonstrating the decreased rate of glycolysis for energy production. Overall, our findings reveal a novel metabolic phenomenon in Canavan disease and NAA metabolism that allows to assign a novel function of N-acetylaspartate.

Interactions between neurons and glia occupy a central role in many aspects of development, maintenance, and function of the central nervous system (CNS). A fundamental event in CNS development is the elaboration of two distinct neuronal processes, axons and dendrites. The overall aim of this research was to characterize the interactions between central nervous system neurons and astroglial cells that regulate dendrite growth from cerebral cortical neurons. Embryonic (E18) mouse cerebral cortical neurons were cocultured with early postnatal (P4) rat astroglia derived from cerebral cortex, retina, olfactory bulb, mesencephalon, striatum and spinal cord. Axon and dendrite outgrowth from isolated neurons was quantified using morphological and double-labeling immunohistochemical techniques at 18 hours and 1, 3 and 5 days in vitro. Neurons initially extended the same number of neurites, regardless of the source of glial monolayer; however, astroglial cells differed in their ability to maintain primary dendrites. Homotypic cortical astroglia maintained the greatest number of primary dendrites. Astroglia derived from the olfactory bulb and retina maintained intermediate numbers of dendrites, whereas only a small number of primary dendrites were maintained by astroglia derived from striatum, spinal cord or mesencephalon. Initially longer axons were observed from neurons grown on astroglia that did not maintain dendrite number. After 5 days in vitro, axon growth was similar on the various monolayers, total primary dendrite outgrowth, however, was nearly threefold greater on astroglia derived from the cortex, retina and olfactory bulb than on astroglia derived from mesencephalon, striatum or spinal cord. This effect was principally on the number of primary dendrites rather than the elongation of individual dendrites and was independent of neuron survival. Similar morphological differences were observed after 5 days in vitro when cortical neurons were grown on polylysine in either a noncontact coculture system where astroglia continuously conditioned the culture medium or in astroglial conditioned medium. Preliminary biochemical analysis of the medium conditioned by cortical astroglia using heat and trypsin degradation, ultracentrifugation, dialysis, and heparin affinity chromatography suggested that a heparin binding protein with a molecular weight between 10 and 100kDa may be responsible for astroglial mediated dendrite growth. Neurons that were grown in medium conditioned by either mesencephalic or cortical astroglia for the first 24 hours followed by culture medium from astroglia of the alternate source for 4 days in vitro, confirmed that astroglia maintained, rather than initiated, the outgrowth of the primary dendritic arbor. In the next series of experiments, E18 mouse cortical neurons were cocultured with neonatal (P4) or mature (P12) rat astroglia derived from cortex and mesencephalon or astroglia derived from P4 and P12 lesioned cortex. After 5 days in vitro, the maturational age of astroglia did not appear to alter the extent of primary dendrite growth; instead dendrite growth reflected the region of the CNS from which the astroglia were derived. By contrast, a reduced ability to support axon growth from mouse cortical neurons in culture was observed on astroglia derived from mature rat cortex or mesencephalon. Reactive astroglia demonstrated similar neurite supporting characteristics to mature astroglia and were able to maintain dendrite growth, principally primary dendrite number. Axon elongation, however, was reduced on both neonatal and mature reactive astroglia. Neuron survival did not correlate with the ability of the various astroglia to support process outgrowth. Collectively these results indicate: 1) neuron-glial interactions are critical for the regulation of process outgrowth from embryonic cortical neurons in vitro, 2) axon and dendrite growth appear to be differently controlled by astroglia, 3) CNS astroglia demonstrate regional differences in maintaining, but not initiating growth of the primary dendritic arbor, 4) this effect may be due, in part, to release of a diffusible heparin binding protein factor, and 5) mature and reactive astroglia support primary dendrite, but limited axon growth. We propose therefore that the local astroglial environment maintains primary dendrite growth from neurons until synaptic contacts can be established. A mechanism that maintains the primary dendritic arbor and allows separate regulation of axon and dendrite growth, prior to the arrival of afferents, may be critical for establishing appropriate and specific synaptic connections. These findings have important implications in understanding development and function of the mammalian central nervous system and may lead to novel strategies for intervention in acute and chronic neurological disorders.

As one of the closest relatives of arthropods, Onychophora plays an important role in understanding the evolution of arthropod body plans. Currently there is controversy surrounding the evolution of the brain among the ecdysozoan clades, which shows a collar-shaped, circumoral organization in cycloneuralians but a ganglionic architecture in panarthropods. Based on the innervation pattern of lip papillae surrounding the mouth, the onychophoran brain has been interpreted as a circumoral ring, suggesting that this organization is an ancestral feature of Ecdysozoa. However, this interpretation is inconsistent with other published data. To explore the evolutionary origin of the onychophoran mouth and to shed light on the evolution of the ecdysozoan brains, we analyzed the innervation pattern and morphogenesis of the oral lip papillae in the onychophoran Euperipatoides rowelli using DNA labeling, immunocytochemistry, and neuronal tracing techniques. Our morphogenetic data revealed that the seven paired and one unpaired oral lip papillae arise from three anterior-most body segments. Retrograde fills show that only the first and the third nerves supplying the lip papillae are associated with cell bodies within the brain, whereas the second nerve exclusively receives fibers from somata of peripheral neurons located in the lip papillae. According to our anterograde fills and immunocytochemical data, the first nerve supplies the anterior-most pair of lip papillae, whereas the second and the third nerves are associated with the second to fifth and second to eighth lip papillae, respectively. These data suggest that the lip papillae of E. rowelli are mainly innervated by the proto- and deutocerebrum, whereas there are only a few additional cell bodies situated posterior to the brain. According to these findings, the overall innervation pattern of the oral lip papillae in E. rowelli is incompatible with the interpretation of the onychophoran brain as a modified circumoral ring.

Newly designed pyrrolo[2,1-c][1,4]benzodiazepines tricyclic skeleton has shown potential clusters of cannabinoid receptors CB1/CB2 selective ligands. CB2 plays a critical role in microglial-derived neuroinflammation, where it modulates cell proliferation, migration, and differentiation into M1 or M2 phenotypes. Beginning with computer-based docking studies accounting the recently discovered X-ray crystal structure of CB2, we designed a series of PBD analogs as potential ligands of CB2 and tested their binding affinities. Interestingly, computational studies and theoretical binding affinities of several selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs, have revealed the presence of potential selectivity in binding attraction toward CB1 and CB2. Reported here is the discovery of the first representatives of this series of selective binding to CB2. Preliminary data showed that this class of molecules display potential binding efficacy toward the cannabinoid receptors tested. Intriguingly, initial cannabinoid binding assay showed a selective binding affinity of 4g and 4h showed Ki of 0.49 and 4.7 μM toward CB2 receptors while no binding was observed to CB1. The designed leads have shown remarkable stability pattern at the physiological pH magnifying their therapeutic values. We hypothesize that the PBD tricyclic structure offers the molecule an appropriate three-dimensional conformation to fit snugly within the active site of CB2 receptors, giving them superiority over the reported CB2 agonists/inverse agonists. Our findings suggested that the attachment of heterocyclic ring through the condensation of diazepine hydrazone and S- or N-heterocyclic aldehydes enhances the selectivity of CB2 over CB1. [Figure not available: see fulltext.].

Psychoneuroimmunology is an interdisciplinary area that examines the interaction between behavior, the central nervous system, and the immune system. Many investigations have utilized a taste aversion paradigm to examine the effects of classical conditioning on an immune response. The procedure generally consists of an animal ingesting a novel flavor, and then being made ill and immunosuppressed by injection of a pharmacological agent. The animal is provided access to that flavor at a later time. The rejection of the novel flavor on the test day is called taste aversion and the depressed antibody titer has been labeled conditioned immunosuppression. The present research was designed condition a secondary immune response and expand the evaluation of such conditioning to include both antibody titer and affinity. The Enzyme Linked Immunoassay was also introduced as the procedure of choice to quantify immune reactivity. A depression in antibody titer and affinity was found following exposure to three of four test trials. Taste aversion did not correlate with the immune response as increased consumption of the novel flavor was exhibited on the third and fourth test trial. In the second experiment, the dosage of cyclophosphamide was increased. A depression in antibody affinity was found after the third and fourth test trials, which was consistent with the results of the first experiment. Unlike the first experiment, a depression in antibody titer was not attained on test days. Although taste aversion was observed in the treatment group on three of the four test trials, it had extinguished by test four. The results support the concept of conditioned suppression of an antigen specific immune response by exposure to the taste aversion paradigm. An important contribution of the present research was the use and modification of a precise and sensitive assay for quantification of titer and affinity; the demonstration of conditioned suppression in both antibody titer and affinity; and the demonstration of conditioned immunosuppression with a single component CS.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20260号 / 医博第4219号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 前川 平, 教授 木原 正博, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM