The Relationship of Aggregate Durability to Trace Element Content, Interim Report, HR-266, 1984

This report is a brief overview of the recent Iowa Department of Transportation research in the area of durability of Portland cement, concrete under the direction of Wendeli Dubberke. Present plans are to publish a more detailed report on low Portland cement concrete- durability research in January, 1985.

Investigation of PCC Pavement Deterioration: A Few Facts are Worth More than 100 Opinions, Interim Report, HR-2074, 1995

Portland Cement Concrete (PCC) pavement has served the State of Iowa well for many years. The oldest Iowa pavement was placed in LeMars in 1904. Beginning in 1931, many miles of PCC pavement were built to "get out of the mud.” Many of these early pavements provided good performance without deterioration for more than 50 years. In the late 1950's, Iowa was faced with severe PCC pavement deterioration referred to as D cracking. Research identified the cause of this deterioration as crushed limestone containing a bad pore system. Selective quarrying and ledge control has alleviated this problem. In 1990, cracking deterioration was identified on a three year old pavement on us 20 in central Iowa. The coarse aggregate was a crushed limestone with an excellent history of performance in PCC pavement. Examination of cores showed very few cracks through the coarse aggregate particles. The cracks were predominately confined to the matrix. The deterioration was identified as alkali-silica reactivity (ASR) by a consultant.

BRAIN DAMAGE IN METHYLMALONIC ACIDURIA: 2-METHYLCITRATE LEADS TO AMMONIA INCREASE AND APOPTOSIS

A 3D in vitro model of rat organotypic brain cell cultures in aggregates was used to investigate neurotoxicity mechanisms in methylmalonic aciduria. 1 mM methylmalonate (MMA), 2-methylcitrate (2-MCA) or propionate (PA) were repeatedly added to the culture media at two different time points of the cultures. In cultures treated with 2-MCA, we observed a significant increase of lactate in the medium, consistent with a possible inhibition of Krebs cycle and respiratory chain, as described earlier in the literature. Interestingly, we further observed that 2-MCA induced an important increase in ammonia production with concomitant decrease of glutamine concentrations, which suggests an inhibition of the astrocytic enzyme glutamine synthetase. These previously unreported findings may uncover a pathogenic mechanism in this disease with deleterious effects on early stages of brain development. By immunohistochemistry we could show that 2-MCA substantially increased the number of apoptotic cells. On the cellular level, 2-MCA had a toxic effect (cell swelling and cell death) on glial cells, but not on neurons. Surprisingly, MMA seemed to have a growth stimulating effect on the cultures. We can conclude that 2-MCA was the most toxic metabolite in our model for methylmalonic aciduria inducing ammonia accumulation and massive apoptosis in brain cells.

Use of aggregating cell cultures for toxicological studies.

Relatively simple techniques are now available which allow the preparation of large quantities of highly reproducible aggregate cultures from fetal rat brain or liver cells, and to grow them in a chemically defined medium. Since these cultures exhibit extensive histotypic cellular reorganization and maturation, they offer unique possibilities for developmental studies. Therefore, the purpose of the present study was to investigate the usefulness of these cultures in developmental toxicology. Aggregating brain cell cultures were exposed at different developmental stages to model drugs (i.e., antimitotic, neurotoxic, and teratogenic agents) and assayed for their responsiveness by measuring a set of biochemical parameters (i.e., total protein and DNA content, cell type-specific enzyme activities) which permit a monitoring of cellular growth and maturation. It was found that each test compound elicited a distinct, dose-dependent response pattern, which may ultimately serve to screen and classify toxic drugs by using mechanistic criteria. In addition, it could be shown that aggregating liver cell cultures are capable of toxic drug activation, and that they can be used in co-culture with brain cell aggregates, providing a potential model for complementary toxicological and metabolic studies.

Intracellular trafficking of interleukin-1 receptor I requires Tollip.

Interleukin-1 receptor (IL-1RI) is a master regulator of inflammation and innate immunity. When triggered by IL-1beta, IL-1RI aggregates with IL-1R-associated protein (IL-1RAcP) and forms a membrane proximal signalosome that potently activates downstream signaling cascades. IL-1beta also rapidly triggers endocytosis of IL-1RI. Although internalization of IL-1RI significantly impacts signaling, very little is known about trafficking of IL-1RI and therefore about precisely how endocytosis modulates the overall cellular response to IL-1beta. Upon internalization, activated receptors are often sorted through endosomes and delivered to lysosomes for degradation. This is a highly regulated process that requires ubiquitination of cargo proteins as well as protein-sorting complexes that specifically recognize ubiquitinated cargo. Here, we show that IL-1beta induces ubiquitination of IL-1RI and that via these attached ubiquitin groups, IL-1RI interacts with the ubiquitin-binding protein Tollip. By using an assay to follow trafficking of IL-1RI from the cell surface to late endosomes and lysosomes, we demonstrate that Tollip is required for sorting of IL-1RI at late endosomes. In Tollip-deficient cells and cells expressing only mutated Tollip (incapable of binding IL-1RI and ubiquitin), IL-1RI accumulates on late endosomes and is not efficiently degraded. Furthermore, we show that IL-1RI interacts with Tom1, an ubiquitin-, clathrin-, and Tollip-binding protein, and that Tom1 knockdown also results in the accumulation of IL-1RI at late endosomes. Our findings suggest that Tollip functions as an endosomal adaptor linking IL-1RI, via Tom1, to the endosomal degradation machinery.

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses.

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.

The Hydrophobic interaction: modeling hydrophobic interactions and aggregation of non-polar particles in aqueous solutions

Malgré son importance dans notre vie de tous les jours, certaines propriétés de l?eau restent inexpliquées. L'étude des interactions entre l'eau et les particules organiques occupe des groupes de recherche dans le monde entier et est loin d'être finie. Dans mon travail j'ai essayé de comprendre, au niveau moléculaire, ces interactions importantes pour la vie. J'ai utilisé pour cela un modèle simple de l'eau pour décrire des solutions aqueuses de différentes particules. Récemment, l?eau liquide a été décrite comme une structure formée d?un réseau aléatoire de liaisons hydrogènes. En introduisant une particule hydrophobe dans cette structure à basse température, certaines liaisons hydrogènes sont détruites ce qui est énergétiquement défavorable. Les molécules d?eau s?arrangent alors autour de cette particule en formant une cage qui permet de récupérer des liaisons hydrogènes (entre molécules d?eau) encore plus fortes : les particules sont alors solubles dans l?eau. A des températures plus élevées, l?agitation thermique des molécules devient importante et brise les liaisons hydrogènes. Maintenant, la dissolution des particules devient énergétiquement défavorable, et les particules se séparent de l?eau en formant des agrégats qui minimisent leur surface exposée à l?eau. Pourtant, à très haute température, les effets entropiques deviennent tellement forts que les particules se mélangent de nouveau avec les molécules d?eau. En utilisant un modèle basé sur ces changements de structure formée par des liaisons hydrogènes j?ai pu reproduire les phénomènes principaux liés à l?hydrophobicité. J?ai trouvé une région de coexistence de deux phases entre les températures critiques inférieure et supérieure de solubilité, dans laquelle les particules hydrophobes s?agrègent. En dehors de cette région, les particules sont dissoutes dans l?eau. J?ai démontré que l?interaction hydrophobe est décrite par un modèle qui prend uniquement en compte les changements de structure de l?eau liquide en présence d?une particule hydrophobe, plutôt que les interactions directes entre les particules. Encouragée par ces résultats prometteurs, j?ai étudié des solutions aqueuses de particules hydrophobes en présence de co-solvants cosmotropiques et chaotropiques. Ce sont des substances qui stabilisent ou déstabilisent les agrégats de particules hydrophobes. La présence de ces substances peut être incluse dans le modèle en décrivant leur effet sur la structure de l?eau. J?ai pu reproduire la concentration élevée de co-solvants chaotropiques dans le voisinage immédiat de la particule, et l?effet inverse dans le cas de co-solvants cosmotropiques. Ce changement de concentration du co-solvant à proximité de particules hydrophobes est la cause principale de son effet sur la solubilité des particules hydrophobes. J?ai démontré que le modèle adapté prédit correctement les effets implicites des co-solvants sur les interactions de plusieurs corps entre les particules hydrophobes. En outre, j?ai étendu le modèle à la description de particules amphiphiles comme des lipides. J?ai trouvé la formation de différents types de micelles en fonction de la distribution des regions hydrophobes à la surface des particules. L?hydrophobicité reste également un sujet controversé en science des protéines. J?ai défini une nouvelle échelle d?hydrophobicité pour les acides aminés qui forment des protéines, basée sur leurs surfaces exposées à l?eau dans des protéines natives. Cette échelle permet une comparaison meilleure entre les expériences et les résultats théoriques. Ainsi, le modèle développé dans mon travail contribue à mieux comprendre les solutions aqueuses de particules hydrophobes. Je pense que les résultats analytiques et numériques obtenus éclaircissent en partie les processus physiques qui sont à la base de l?interaction hydrophobe.<br/><br/>Despite the importance of water in our daily lives, some of its properties remain unexplained. Indeed, the interactions of water with organic particles are investigated in research groups all over the world, but controversy still surrounds many aspects of their description. In my work I have tried to understand these interactions on a molecular level using both analytical and numerical methods. Recent investigations describe liquid water as random network formed by hydrogen bonds. The insertion of a hydrophobic particle at low temperature breaks some of the hydrogen bonds, which is energetically unfavorable. The water molecules, however, rearrange in a cage-like structure around the solute particle. Even stronger hydrogen bonds are formed between water molecules, and thus the solute particles are soluble. At higher temperatures, this strict ordering is disrupted by thermal movements, and the solution of particles becomes unfavorable. They minimize their exposed surface to water by aggregating. At even higher temperatures, entropy effects become dominant and water and solute particles mix again. Using a model based on these changes in water structure I have reproduced the essential phenomena connected to hydrophobicity. These include an upper and a lower critical solution temperature, which define temperature and density ranges in which aggregation occurs. Outside of this region the solute particles are soluble in water. Because I was able to demonstrate that the simple mixture model contains implicitly many-body interactions between the solute molecules, I feel that the study contributes to an important advance in the qualitative understanding of the hydrophobic effect. I have also studied the aggregation of hydrophobic particles in aqueous solutions in the presence of cosolvents. Here I have demonstrated that the important features of the destabilizing effect of chaotropic cosolvents on hydrophobic aggregates may be described within the same two-state model, with adaptations to focus on the ability of such substances to alter the structure of water. The relevant phenomena include a significant enhancement of the solubility of non-polar solute particles and preferential binding of chaotropic substances to solute molecules. In a similar fashion, I have analyzed the stabilizing effect of kosmotropic cosolvents in these solutions. Including the ability of kosmotropic substances to enhance the structure of liquid water, leads to reduced solubility, larger aggregation regime and the preferential exclusion of the cosolvent from the hydration shell of hydrophobic solute particles. I have further adapted the MLG model to include the solvation of amphiphilic solute particles in water, by allowing different distributions of hydrophobic regions at the molecular surface, I have found aggregation of the amphiphiles, and formation of various types of micelle as a function of the hydrophobicity pattern. I have demonstrated that certain features of micelle formation may be reproduced by the adapted model to describe alterations of water structure near different surface regions of the dissolved amphiphiles. Hydrophobicity remains a controversial quantity also in protein science. Based on the surface exposure of the 20 amino-acids in native proteins I have defined the a new hydrophobicity scale, which may lead to an improvement in the comparison of experimental data with the results from theoretical HP models. Overall, I have shown that the primary features of the hydrophobic interaction in aqueous solutions may be captured within a model which focuses on alterations in water structure around non-polar solute particles. The results obtained within this model may illuminate the processes underlying the hydrophobic interaction.<br/><br/>La vie sur notre planète a commencé dans l'eau et ne pourrait pas exister en son absence : les cellules des animaux et des plantes contiennent jusqu'à 95% d'eau. Malgré son importance dans notre vie de tous les jours, certaines propriétés de l?eau restent inexpliquées. En particulier, l'étude des interactions entre l'eau et les particules organiques occupe des groupes de recherche dans le monde entier et est loin d'être finie. Dans mon travail j'ai essayé de comprendre, au niveau moléculaire, ces interactions importantes pour la vie. J'ai utilisé pour cela un modèle simple de l'eau pour décrire des solutions aqueuses de différentes particules. Bien que l?eau soit généralement un bon solvant, un grand groupe de molécules, appelées molécules hydrophobes (du grecque "hydro"="eau" et "phobia"="peur"), n'est pas facilement soluble dans l'eau. Ces particules hydrophobes essayent d'éviter le contact avec l'eau, et forment donc un agrégat pour minimiser leur surface exposée à l'eau. Cette force entre les particules est appelée interaction hydrophobe, et les mécanismes physiques qui conduisent à ces interactions ne sont pas bien compris à l'heure actuelle. Dans mon étude j'ai décrit l'effet des particules hydrophobes sur l'eau liquide. L'objectif était d'éclaircir le mécanisme de l'interaction hydrophobe qui est fondamentale pour la formation des membranes et le fonctionnement des processus biologiques dans notre corps. Récemment, l'eau liquide a été décrite comme un réseau aléatoire formé par des liaisons hydrogènes. En introduisant une particule hydrophobe dans cette structure, certaines liaisons hydrogènes sont détruites tandis que les molécules d'eau s'arrangent autour de cette particule en formant une cage qui permet de récupérer des liaisons hydrogènes (entre molécules d?eau) encore plus fortes : les particules sont alors solubles dans l'eau. A des températures plus élevées, l?agitation thermique des molécules devient importante et brise la structure de cage autour des particules hydrophobes. Maintenant, la dissolution des particules devient défavorable, et les particules se séparent de l'eau en formant deux phases. A très haute température, les mouvements thermiques dans le système deviennent tellement forts que les particules se mélangent de nouveau avec les molécules d'eau. A l'aide d'un modèle qui décrit le système en termes de restructuration dans l'eau liquide, j'ai réussi à reproduire les phénomènes physiques liés à l?hydrophobicité. J'ai démontré que les interactions hydrophobes entre plusieurs particules peuvent être exprimées dans un modèle qui prend uniquement en compte les liaisons hydrogènes entre les molécules d'eau. Encouragée par ces résultats prometteurs, j'ai inclus dans mon modèle des substances fréquemment utilisées pour stabiliser ou déstabiliser des solutions aqueuses de particules hydrophobes. J'ai réussi à reproduire les effets dûs à la présence de ces substances. De plus, j'ai pu décrire la formation de micelles par des particules amphiphiles comme des lipides dont la surface est partiellement hydrophobe et partiellement hydrophile ("hydro-phile"="aime l'eau"), ainsi que le repliement des protéines dû à l'hydrophobicité, qui garantit le fonctionnement correct des processus biologiques de notre corps. Dans mes études futures je poursuivrai l'étude des solutions aqueuses de différentes particules en utilisant les techniques acquises pendant mon travail de thèse, et en essayant de comprendre les propriétés physiques du liquide le plus important pour notre vie : l'eau.

Engineered Trx2p industrial yeast strain protects glycolysis and fermentation proteins from oxidative carbonylation during biomass propagation

Background: In the yeast biomass production process, protein carbonylation has severe adverse effects since it diminishes biomass yield and profitability of industrial production plants. However, this significant detriment of yeast performance can be alleviated by increasing thioredoxins levels. Thioredoxins are important antioxidant defenses implicated in many functions in cells, and their primordial functions include scavenging of reactive oxygen species that produce dramatic and irreversible alterations such as protein carbonylation. Results: In this work we have found several proteins specifically protected by yeast Thioredoxin 2 (Trx2p). Bidimensional electrophoresis and carbonylated protein identification from TRX-deficient and TRX-overexpressing cells revealed that glycolysis and fermentation-related proteins are specific targets of Trx2p protection. Indeed, the TRX2 overexpressing strain presented increased activity of the central carbon metabolism enzymes. Interestingly, Trx2p specifically preserved alcohol dehydrogenase I (Adh1p) from carbonylation, decreased oligomer aggregates and increased its enzymatic activity. Conclusions: The identified proteins suggest that the fermentative capacity detriment observed under industrial conditions in T73 wine commercial strain results from the oxidative carbonylation of specific glycolytic and fermentation enzymes. Indeed, increased thioredoxin levels enhance the performance of key fermentation enzymes such as Adh1p, which consequently increases fermentative capacity.

The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases.

Astrocyte reactivity is a hallmark of neurodegenerative diseases (ND), but its effects on disease outcomes remain highly debated. Elucidation of the signaling cascades inducing reactivity in astrocytes during ND would help characterize the function of these cells and identify novel molecular targets to modulate disease progression. The Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is associated with reactive astrocytes in models of acute injury, but it is unknown whether this pathway is directly responsible for astrocyte reactivity in progressive pathological conditions such as ND. In this study, we examined whether the JAK/STAT3 pathway promotes astrocyte reactivity in several animal models of ND. The JAK/STAT3 pathway was activated in reactive astrocytes in two transgenic mouse models of Alzheimer's disease and in a mouse and a nonhuman primate lentiviral vector-based model of Huntington's disease (HD). To determine whether this cascade was instrumental for astrocyte reactivity, we used a lentiviral vector that specifically targets astrocytes in vivo to overexpress the endogenous inhibitor of the JAK/STAT3 pathway [suppressor of cytokine signaling 3 (SOCS3)]. SOCS3 significantly inhibited this pathway in astrocytes, prevented astrocyte reactivity, and decreased microglial activation in models of both diseases. Inhibition of the JAK/STAT3 pathway within reactive astrocytes also increased the number of huntingtin aggregates, a neuropathological hallmark of HD, but did not influence neuronal death. Our data demonstrate that the JAK/STAT3 pathway is a common mediator of astrocyte reactivity that is highly conserved between disease states, species, and brain regions. This universal signaling cascade represents a potent target to study the role of reactive astrocytes in ND.

Approches de gene silencing pour le traitement de la maladie de Huntington [Gene silencing approaches for the treatment of Huntington's disease].

Huntington's disease is a rare neurodegenerative disease caused by a pathologic CAG expansion in the exon 1 of the huntingtin (HTT) gene. Aggregation and abnormal function of the mutant HTT (mHTT) cause motor, cognitive and psychiatric symptoms in patients, which lead to death in 15-20 years. Currently, there is no treatment for HD. Experimental approaches based on drug, cell or gene therapy are developed and reach progressively to the clinic. Among them, mHTT silencing using small non-coding nucleic acids display important physiopathological benefit in HD experimental models.

NLRP3 Inflammasome Is Expressed and Functional in Mouse Brain Microglia but Not in Astrocytes.

Neuroinflammation is the local reaction of the brain to infection, trauma, toxic molecules or protein aggregates. The brain resident macrophages, microglia, are able to trigger an appropriate response involving secretion of cytokines and chemokines, resulting in the activation of astrocytes and recruitment of peripheral immune cells. IL-1β plays an important role in this response; yet its production and mode of action in the brain are not fully understood and its precise implication in neurodegenerative diseases needs further characterization. Our results indicate that the capacity to form a functional NLRP3 inflammasome and secretion of IL-1β is limited to the microglial compartment in the mouse brain. We were not able to observe IL-1β secretion from astrocytes, nor do they express all NLRP3 inflammasome components. Microglia were able to produce IL-1β in response to different classical inflammasome activators, such as ATP, Nigericin or Alum. Similarly, microglia secreted IL-18 and IL-1α, two other inflammasome-linked pro-inflammatory factors. Cell stimulation with α-synuclein, a neurodegenerative disease-related peptide, did not result in the release of active IL-1β by microglia, despite a weak pro-inflammatory effect. Amyloid-β peptides were able to activate the NLRP3 inflammasome in microglia and IL-1β secretion occurred in a P2X7 receptor-independent manner. Thus microglia-dependent inflammasome activation can play an important role in the brain and especially in neuroinflammatory conditions.

Methylglyoxal Produced by Amyloid- Peptide-Induced Nitrotyrosination of Triosephosphate Isomerase Triggers Neuronal Death in Alzheimer’s Disease

Amyloid-β peptide (Aβ) aggregates induce nitro-oxidative stress, contributing to the characteristic neurodegeneration found in Alzheimer's disease (AD). One of the most strongly nitrotyrosinated proteins in AD is the triosephosphate isomerase (TPI) enzyme which regulates glycolytic flow, and its efficiency decreased when it is nitrotyrosinated. The main aims of this study were to analyze the impact of TPI nitrotyrosination on cell viability and to identify the mechanism behind this effect. In human neuroblastoma cells (SH-SY5Y), we evaluated the effects of Aβ42 oligomers on TPI nitrotyrosination. We found an increased production of methylglyoxal (MG), a toxic byproduct of the inefficient nitro-TPI function. The proapoptotic effects of Aβ42 oligomers, such as decreasing the protective Bcl2 and increasing the proapoptotic caspase-3 and Bax, were prevented with a MG chelator. Moreover, we used a double mutant TPI (Y165F and Y209F) to mimic nitrosative modifications due to Aβ action. Neuroblastoma cells transfected with the double mutant TPI consistently triggered MG production and a decrease in cell viability due to apoptotic mechanisms. Our data show for the first time that MG is playing a key role in the neuronal death induced by Aβ oligomers. This occurs because of TPI nitrotyrosination, which affects both tyrosines associated with the catalytic center.

Ligand binding to heparan sulfate proteoglycans induces their aggregation and distribution along actin cytoskeleton

Cell surface heparan sulfate proteoglycans (HSPGs) participate in molecular events that regulate cell adhesion, migration, and proliferation. The present study demonstrates that soluble heparin-binding proteins or cross-linking antibodies induce the aggregation of cell surface HSPGs and their distribution along underlying actin filaments. Immunofluorescence and confocal microscopy and immunogold and electron microscopy indicate that, in the absence of ligands, HSPGs are irregularly distributed on the fibroblast cell surface, without any apparent codistribution with the actin cytoskeleton. In the presence of ligand (lipoprotein lipase) or antibodies against heparan sulfate, HSPGs aggregate and colocalize with the actin cytoskeleton. Triton X-100 extraction and immunoelectron microscopy have demonstrated that in this condition HSPGs were clustered and associated with the actin filaments. Crosslinking experiments that use biotinylated lipoprotein lipase have revealed three major proteoglycans as binding sites at the fibroblast cell surface. These cross-linked proteoglycans appeared in the Triton X-100 insoluble fraction. Platinum/carbon replicas of the fibroblast surface incubated either with lipoprotein lipase or antiheparan sulfate showed large aggregates of HSPGs regularly distributed along cytoplasmic fibers. Quantification of the spacing between HSPGs by confocal microscopy confirmed that the nonrandom distribution of HSPG aggregates along the actin cytoskeleton was induced by ligand binding. When cells were incubated either with lipoprotein lipase or antibodies against heparan sulfate, the distance between immunofluorescence spots was uniform. In contrast, the spacing between HSPGs on fixed cells not incubated with ligand was more variable. This highly organized spatial relationship between actin and proteoglycans suggests that cortical actin filaments could organize the molecular machinery involved in signal transduction and molecular movements on the cell surface that are triggered by heparin-binding proteins.

Creatine deficiency syndomes : a new model of partial GAMT deficiency affecting brain cell development

Les syndromes de déficiences cérébrales en créatine (CCDS) sont dus à des mutations dans les gènes GATM et G AMT (codant pour les enzymes AGAT et G AMT de la voie de synthèse de créatine) ainsi que SLC6A8 (transporteur de créatine), et génèrent une absence ou une très forte baisse de créatine (Cr) dans le cerveau, mesurée par spectroscopic de résonance magnétique. Les patients CCDS développent des handicaps neurologiques sévères. Les patients AGAT et GAMT peuvent être traités avec des doses importantes de Cr, mais gardent dans la plupart des cas des séquelles neurologiques irréversibles. Aucun traitement efficace n'existe à ce jour pour la déficience en SLC6A8. Bien que de nombreux modèles aient été développés pour comprendre la Cr cérébrale en conditions physiologiques, les pathomécanismes des CCDS ne sont pas encore compris. Des souris transgéniques pour les gènes Gatm, Gamt et Slc6a8 ont été générées, mais elles ne miment que partiellement la pathologie humaine. Parmi les CCDS, la déficience en GAMT est la plus sévère, en raison de l'accumulation cérébrale de l'intermédiaire guanidinoacétate (GAA). Alors que la toxicité cérébrale du GAA a été étudiée par exposition directe au GAA d'animaux adultes sains, les mécanismes de la toxicité du GAA en condition de déficience en GAMT dans le cerveau en développement sont encore inconnus. Le but de ce projet était donc de développer un modèle de déficience en GAMT dans des cultures 3D primaires de cellules nerveuses de rat en agrégats par knock-down du gène GAMT, en utilisant un virus adéno-associé (AAV) induisant le mécanisme d'interférence à l'ARN (RNAi). Le virus scAAV2, à la multiplicité d'infection de 1000, s'est révélé le plus efficace pour transduire tous les types de cellules nerveuses des cultures (neurones, astrocytes, oligodendrocytes), et générer un knock-down maximal de la protéine GAMT de 85% (jour in vitro 18). Cette déficience partielle en GAMT s'est révélée insuffisante pour générer une déficience en Cr, mais a causé l'accumulation attendue de GAA, à des doses comparables aux niveaux observés dans le LCR des patients GAMT. Le GAA a induit une croissance axonale anarchique accompagnée d'une baisse de l'apoptose naturelle, suivis par une induction tardive de mort cellulaire non-apoptotique. Le co-traitement par la Cr a prévenu tous les effets toxiques du GAA. Ce travail montre que l'accumulation de GAA en absence de déficience en Cr est suffisante pour affecter le développement du tissu nerveux, et suggère que des formes de déficiences en GAMT supplémentaires, ne présentant pas de déficiences en Cr, pourraient être découvertes par mesure du GAA, en particulier à travers les programmes récemment proposés de dépistage néonatal de la déficience en GAMT. -- Cerebral creatine deficiency syndromes (CCDS) are caused by mutations in the genes GATM and GAMT (respectively coding for the two enzymes of the creatine synthetic pathway, AGAT and GAMT) as well as SLC6A8 (creatine transporter), and lead to the absence or very strong decrease of creatine (Cr) in the brain when measured by magnetic resonance spectroscopy. Affected patients show severe neurological impairments. While AGAT and GAMT deficient patients can be treated with high dosages of Cr, most remain with irreversible brain sequelae. No treatment has been successful so far for SLC6A8 deficiency. While many models have helped understanding the cerebral Cr pathways in physiological conditions, the pathomechanisms underlying CCDS are yet to be elucidated. Transgenic mice carrying mutations in the Gatm, Gamt and Slc6a8 genes have been developed, but only partially mimic the human pathology. Among CCDS, GAMT deficiency is the most severe, due to the CNS accumulation of the guanidinoacetate (GAA) intermediate. While brain toxicity of GAA has been explored through direct GAA exposure of adult healthy animals, the mechanisms underlying GAA toxicity in GAMT deficiency conditions on the developing CNS are yet unknown. The aim of this project was thus to develop and characterize a GAMT deficiency model in developing brain cells by gene knockdown, by adeno-associated virus (AAV)-driven RNA interference (RNAi) in rat 3D organotypic primary brain cell cultures in aggregates. scAAV2 with a multiplicity of infection of 1000 was shown as the most efficient serotype, was able to transduce all brain cell types (neurons, astrocytes, oligodendrocytes) and to induce a maximal GAMT protein knockdown of 85% (day in vitro 18). Metabolite analysis showed that partial GAMT knockdown was insufficient to induce Cr deficiency but generated the awaited GAA accumulation at concentrations comparable to the levels observed in cerebrospinal fluid of GAMT-deficient patients. Accumulated GAA induced axonal hypersprouting paralleled with inhibition of natural apoptosis, followed by a later induction in non-apoptotic cell death. Cr supplementation led to the prevention of all GAA-induced toxic effects. This work shows that GAA accumulation without Cr deficiency is sufficient to affect CNS development, and suggests that additional partial GAMT deficiencies, which may not show the classical brain Cr deficiency, may be discovered through GAA measurement including by recently proposed neonatal screening programs for GAMT deficiency.

Emergence of assortative mixing between clusters of cultured neurons

The analysis of the activity of neuronal cultures is considered to be a good proxy of the functional connectivity of in vivo neuronal tissues. Thus, the functional complex network inferred from activity patterns is a promising way to unravel the interplay between structure and functionality of neuronal systems. Here, we monitor the spontaneous self-sustained dynamics in neuronal cultures formed by interconnected aggregates of neurons (clusters). Dynamics is characterized by the fast activation of groups of clusters in sequences termed bursts. The analysis of the time delays between clusters' activations within the bursts allows the reconstruction of the directed functional connectivity of the network. We propose a method to statistically infer this connectivity and analyze the resulting properties of the associated complex networks. Surprisingly enough, in contrast to what has been reported for many biological networks, the clustered neuronal cultures present assortative mixing connectivity values, meaning that there is a preference for clusters to link to other clusters that share similar functional connectivity, as well as a rich-club core, which shapes a"connectivity backbone" in the network. These results point out that the grouping of neurons and the assortative connectivity between clusters are intrinsic survival mechanisms of the culture.