Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

Synthesis and characterization of C3-symmetric 1,3,5 - Benzenetricarboxamide derived supramolecular systems

The aim of this work was to study the self-assembly process of C3-symmetric molecules. To accomplish this objective 1,3,5 – benzentricarboxamides (BTA) derivatives were obtained. Five C3-symmetric molecules were synthesized in moderate to good yields (39-72%) using azo-benzene, aniline, benzylamine, tryptophan and tyrosine. The aggregation behavior of the BTA derivatives was probed with 1H-NMR spectroscopy, 1H-1H 2D Nuclear Overhauser Effect Spectroscopy (NOESY) and Diffusion Ordered Spectroscopy (DOSY). These experiments allowed to study the influence of H-bonding groups, aromatic rings, unsaturated bonds and the overall geometry in the molecular self-assembly associated with the different structural patterns present on these molecules. The stacking and large molecule behavior where observed in BTA 1, aniline derivative, BTA 4, tyrosine derivative or BTA 5, tryptophan derivative, with several of those discussed functional groups such as unsaturated bonds and H-bonding groups. BTA 5 was used in a few preliminary interaction studies with glucose and ammonium chloride showing interaction with the ammonium ion.

Pentacyclic triterpenes and steroids from the stem bark of uchi (Sacoglottis uchi, Humiriaceae)

The ethanol extract from stem bark of Sacoglottis uchi Huber (popularly known as “uchi” in the Amazon Region) was submitted to chromatographic fractionation. The dichloromethane fractions provided the pentacyclic triterpene 3-oxo-friedelin (1). The dichloromethane:methanol fractions provided the pentacyclic triterpenes pseudotaraxasterol (2), lupeol (3), a-amyrin (4), betulin (5), and methyl 2ß,3ß-dihydroxy-urs-12-en-28-oate (6) and a mixture of the steroids sitosterol (7) and stigmasterol (8). Their chemical structures were determined by NMR spectroscopy and comparison with spectroscopic data from the literature. All compounds are described for the first time in this species.

Caffeine consumption attenuates neurochemical modifications in the hippocampus of streptozotocin-induced diabetic rats.

Type 1 diabetes can affect hippocampal function triggering cognitive impairment through unknown mechanisms. Caffeine consumption prevents hippocampal degeneration and memory dysfunction upon different insults and is also known to affect peripheral glucose metabolism. Thus we now characterized glucose transport and the neurochemical profile in the hippocampus of streptozotocin-induced diabetic rats using in vivo(1)H NMR spectroscopy and tested the effect of caffeine consumption thereupon. We found that hippocampal glucose content and transport were unaltered in diabetic rats, irrespective of caffeine consumption. However diabetic rats displayed alterations in their hippocampal neurochemical profile, which were normalized upon restoration of normoglycaemia, with the exception of myo-inositol that remained increased (36 +/- 5%, p < 0.01 compared to controls) likely reflecting osmolarity deregulation. Compared to controls, caffeine-consuming diabetic rats displayed increased hippocampal levels of myo-inositol (15 +/- 5%, p < 0.05) and taurine (23 +/- 4%, p < 0.01), supporting the ability of caffeine to control osmoregulation. Compared to controls, the hippocampus of diabetic rats displayed a reduced density of synaptic proteins syntaxin, synaptophysin and synaptosome-associated protein of 25 kDa (in average 18 +/- 1%, p < 0.05) as well increased glial fibrillary acidic protein (20 +/- 5%, p < 0.05), suggesting synaptic degeneration and astrogliosis, which were prevented by caffeine consumption. In conclusion, neurochemical alterations in the hippocampus of diabetic rats are not related to defects of glucose transport but likely reflect osmoregulatory adaptations caused by hyperglycemia. Furthermore, caffeine consumption affected this neurochemical adaptation to high glucose levels, which may contribute to its potential neuroprotective effects, namely preventing synaptic degeneration and astrogliosis.

Quantification of in vivo short echo-time proton magnetic resonance spectra at 14.1 T using two different approaches of modelling the macromolecule spectrum

Reliable quantification of the macromolecule signals in short echo-time H-1 MRS spectra is particularly important at high magnetic fields for an accurate quantification of metabolite concentrations (the neurochemical profile) due to effectively increased spectral resolution of the macromolecule components. The purpose of the present study was to assess two approaches of quantification, which take the contribution of macromolecules into account in the quantification step. H-1 spectra were acquired on a 14.1 T/26 cm horizontal scanner on five rats using the ultra-short echo-time SPECIAL (spin echo full intensity acquired localization) spectroscopy sequence. Metabolite concentrations were estimated using LCModel, combined with a simulated basis set of metabolites using published spectral parameters and either the spectrum of macromolecules measured in vivo, using an inversion recovery technique, or baseline simulated by the built-in spline function. The fitted spline function resulted in a smooth approximation of the in vivo macromolecules, but in accordance with previous studies using Subtract-QUEST could not reproduce completely all features of the in vivo spectrum of macromolecules at 14.1 T. As a consequence, the measured macromolecular 'baseline' led to a more accurate and reliable quantification at higher field strengths.

Direct in vivo measurement of glycine and the neurochemical profile in the rat medulla oblongata.

The medulla oblongata (MO) contains a high density of glycinergic synapses and a particularly high concentration of glycine. The aims of this study were to measure directly in vivo the neurochemical profile, including glycine, in MO using a spin-echo-based (1)H MRS sequence at TE?=?2.8 ms and to compare it with three other brain regions (cortex, striatum and hippocampus) in the rat. Glycine was quantified in MO at TE?=?2.8 ms with a Cramér-Rao lower bound (CRLB) of approximately 5%. As a result of the relatively low level of glycine in the other three regions, the measurement of glycine was performed at TE?=?20 ms, which provides a favorable J-modulation of overlapping myo-inositol resonance. The other 14 metabolites composing the neurochemical profile were quantified in vivo in MO with CRLBs below 25%. Absolute concentrations of metabolites in MO, such as glutamate, glutamine, ?-aminobutyrate, taurine and glycine, were in the range of previous in vitro quantifications in tissue extracts. Compared with the other regions, MO had a three-fold higher glycine concentration, and was characterised by reduced (p?<?0.001) concentrations of glutamate (-50?±?4%), glutamine (-54?±?3%) and taurine (-78?±?3%). This study suggests that the functional specialisation of distinct brain regions is reflected in the neurochemical profile.

Neuroprotective role of lactate after cerebral ischemia.

It is well established that lactate can be used as an energy substrate by the brain by conversion to pyruvate and a subsequent oxidation in the mitochondria. Knowing the need for readily metabolizable substrates directly after ischemia and the protective effect of lactate after excitotoxicity, the aim of this study was to investigate whether lactate administration directly after ischemia could be neuroprotective. In vitro, the addition of 4 mmol/L L-lactate to the medium of rat organotypic hippocampal slices, directly after oxygen and glucose deprivation (OGD), protected against neuronal death, whereas a higher dose of 20 mmol/L was toxic. In vivo, after middle cerebral artery occlusion in the mouse, an intracerebroventricular injection of 2 microL of 100 mmol/L L-lactate, immediately after reperfusion, led to a significant decrease in lesion size, which was more pronounced in the striatum, and an improvement in neurologic outcome. A later injection 1 h after reperfusion did not reduce lesion size, but significantly improved neurologic outcome, which is an important point in the context of a potential clinical application. Therefore, a moderate increase in lactate after ischemia may be a therapeutic tool.

α-N-acetylglucosamine-linked O-glycans of sialoglycoproteins (Tc-mucins) from Trypanosoma cruzi Colombiana strain

Trypanosoma cruzi sialoglycoproteins (Tc-mucins) are mucin-like molecules linked to a parasite membrane via a glycosylphosphatidylinositol anchor. We previously determined the structures of Tc-mucin O-glycan domains from several T. cruzi strains and observed significant differences among them. We now report the amino acid content and structure of Tc-mucin O-glycan chains from T. cruzi Colombiana, a strain resistant to common trypanocidal drugs. Amino acid analysis demonstrated the predominance of threonine residues (42%) and helped to identify the O-glycans as belonging to a Tc-mucin family that contain a ²-galactofuranose (²-Galf) residue attached to an α-N-acetylglucosamine (α-GlcNAc) O-4, with the most complex glycan, a pentasaccharide-GlcNAc-ol with a branched trigalactopyranose chain, on the GlcNAc O-6. The presence of ²-Galf on O-glycans from T. cruzi Colombiana mucins supports the use of glycosylation as a phylogenetic marker for the classification of Colombiana in the T. cruzi I group.

In vivo quantification of neuro-glial metabolism and glial glutamate concentration using 1H-[13C] MRS at 14.1T.

Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo (13) C NMR spectroscopy coupled with the infusion labeled glial-specific substrate, such as acetate. In this study, we infused alpha-chloralose anesthetized rats with [2-(13) C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using (1) H-[(13) C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two-compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (Vg ) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (VNT ) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (VPC ) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (Vx (g) and Vx (n) ) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations. In this (13) C-acetate labeling study, we propose a refined two-compartment analysis of brain energy metabolism based on (13) C turnover curves of acetate, glutamate and glutamine measured with state of the art in vivo dynamic MRS at high magnetic field in rats, enabling a deeper understanding of the specific role of glial cells in brain oxidative metabolism. In addition, the robustness of the metabolic fluxes determination relative to MRS data quality was carefully studied.

Steady-state brain glucose transport kinetics re-evaluated with a four-state conformational model.

Glucose supply from blood to brain occurs through facilitative transporter proteins. A near linear relation between brain and plasma glucose has been experimentally determined and described by a reversible model of enzyme kinetics. A conformational four-state exchange model accounting for trans-acceleration and asymmetry of the carrier was included in a recently developed multi-compartmental model of glucose transport. Based on this model, we demonstrate that brain glucose (G(brain)) as function of plasma glucose (G(plasma)) can be described by a single analytical equation namely comprising three kinetic compartments: blood, endothelial cells and brain. Transport was described by four parameters: apparent half saturation constant K(t), apparent maximum rate constant T(max), glucose consumption rate CMR(glc), and the iso-inhibition constant K(ii) that suggests G(brain) as inhibitor of the isomerisation of the unloaded carrier. Previous published data, where G(brain) was quantified as a function of plasma glucose by either biochemical methods or NMR spectroscopy, were used to determine the aforementioned kinetic parameters. Glucose transport was characterized by K(t) ranging from 1.5 to 3.5 mM, T(max)/CMR(glc) from 4.6 to 5.6, and K(ii) from 51 to 149 mM. It was noteworthy that K(t) was on the order of a few mM, as previously determined from the reversible model. The conformational four-state exchange model of glucose transport into the brain includes both efflux and transport inhibition by G(brain), predicting that G(brain) eventually approaches a maximum concentration. However, since K(ii) largely exceeds G(plasma), iso-inhibition is unlikely to be of substantial importance for plasma glucose below 25 mM. As a consequence, the reversible model can account for most experimental observations under euglycaemia and moderate cases of hypo- and hyperglycaemia.

Metabolic and structural rearrangement during dark-induced autophagy in soybean (Glycine max L.) nodules: an electron microscopy and 31P and 13C nuclear magnetic resonance study.

The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using (13)C- and (31)P-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N(2) fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.

Synthesis and characterization of kraft lignin-based epoxy resins

Epoxidization is an interesting way to develop a new application of lignin and therefore to improve its application potential. In this work, kraft lignin-based epoxy resins were obtained by the epoxidization reaction, using the kraft lignin recovered directly from pulping liquor and modified by a methylolation reaction. The methylolated lignins were obtained by the reaction of original kraft lignin with formaldehyde and glyoxal, which is a less volatile and less toxic aldehyde. 1H-NMR spectroscopy showed that methylolated kraft lignin has more hydroxymethyl groups than glyoxalated kraft lignin. For the epoxidization reaction we studied the influence of the lignin:NaOH (w/w) ratio, temperature, and time of the reaction on the properties of the prepared epoxidized lignins. The structures of lignin-based epoxy resins were followed by epoxy index test and FTIR spectroscopy. Optimal conditions were obtained for lignin-based epoxy resin produced at lignin/NaOH = 1/3 at 70 ºC for 3h. Thermogravimetry analysis (TGA) revealed that the epoxidization enhances the thermal stability of lignins and may allow a wider temperature range for applications with lignin epoxy-PF blends

Deep thiopental anesthesia alters steady-state glucose homeostasis but not the neurochemical profile of rat cortex.

Barbiturates are regularly used as an anesthetic for animal experimentation and clinical procedures and are frequently provided with solubilizing compounds, such as ethanol and propylene glycol, which have been reported to affect brain function and, in the case of (1)H NMR experiments, originate undesired resonances in spectra affecting the quantification. As an alternative, thiopental can be administrated without any solubilizing agents. The aim of the study was to investigate the effect of deep thiopental anesthesia on the neurochemical profile consisting of 19 metabolites and on glucose transport kinetics in vivo in rat cortex compared with alpha-chloralose using localized (1)H NMR spectroscopy. Thiopental was devoid of effects on the neurochemical profile, except for the elevated glucose at a given plasma glucose level resulting from thiopental-induced depression of glucose consumption at isoelectrical condition. Over the entire range of plasma glucose levels, steady-state glucose concentrations were increased on average by 48% +/- 8%, implying that an effect of deep thiopental anesthesia on the transport rate relative to cerebral glucose consumption ratio was increased by 47% +/- 8% compared with light alpha-chloralose-anesthetized rats. We conclude that the thiopental-induced isoelectrical condition in rat cortex significantly affected glucose contents by depressing brain metabolism, which remained substantial at isoelectricity.

Non-invasive quantification of brain glycogen absolute concentration.

The only currently available method to measure brain glycogen in vivo is 13C NMR spectroscopy. Incorporation of 13C-labeled glucose (Glc) is necessary to allow glycogen measurement, but might be affected by turnover changes. Our aim was to measure glycogen absolute concentration in the rat brain by eliminating label turnover as variable. The approach is based on establishing an increased, constant 13C isotopic enrichment (IE). 13C-Glc infusion is then performed at the IE of brain glycogen. As glycogen IE cannot be assessed in vivo, we validated that it can be inferred from that of N-acetyl-aspartate IE in vivo: After [1-13C]-Glc ingestion, glycogen IE was 2.2 +/- 0.1 fold that of N-acetyl-aspartate (n = 11, R(2) = 0.77). After subsequent Glc infusion, glycogen IE equaled brain Glc IE (n = 6, paired t-test, p = 0.37), implying isotopic steady-state achievement and complete turnover of the glycogen molecule. Glycogen concentration measured in vivo by 13C NMR (mean +/- SD: 5.8 +/- 0.7 micromol/g) was in excellent agreement with that in vitro (6.4 +/- 0.6 micromol/g, n = 5). When insulin was administered, the stability of glycogen concentration was analogous to previous biochemical measurements implying that glycogen turnover is activated by insulin. We conclude that the entire glycogen molecule is turned over and that insulin activates glycogen turnover.

Deep thiopental anesthesia alters steady-state glucose homeostasis but not the neurochemical profile of rat cortex.

Barbiturates are regularly used as an anesthetic for animal experimentation and clinical procedures and are frequently provided with solubilizing compounds, such as ethanol and propylene glycol, which have been reported to affect brain function and, in the case of (1)H NMR experiments, originate undesired resonances in spectra affecting the quantification. As an alternative, thiopental can be administrated without any solubilizing agents. The aim of the study was to investigate the effect of deep thiopental anesthesia on the neurochemical profile consisting of 19 metabolites and on glucose transport kinetics in vivo in rat cortex compared with alpha-chloralose using localized (1)H NMR spectroscopy. Thiopental was devoid of effects on the neurochemical profile, except for the elevated glucose at a given plasma glucose level resulting from thiopental-induced depression of glucose consumption at isoelectrical condition. Over the entire range of plasma glucose levels, steady-state glucose concentrations were increased on average by 48% +/- 8%, implying that an effect of deep thiopental anesthesia on the transport rate relative to cerebral glucose consumption ratio was increased by 47% +/- 8% compared with light alpha-chloralose-anesthetized rats. We conclude that the thiopental-induced isoelectrical condition in rat cortex significantly affected glucose contents by depressing brain metabolism, which remained substantial at isoelectricity.