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.].

Les gliomes sont les tumeurs cérébrales de l’adulte les plus fréquentes, dont l’étiologie reste encore largement inconnue. Plusieurs études épidémiologiques ont montré l’existence d’une corrélation entre maladies neurodégénératives et cancers cérébraux. Nous avons émis l’hypothèse qu’il existait des dénominateurs moléculaires communs entre ces pathologies. Je me suis particulièrement intéressé au rôle de la parkine (PK), une ubiquitine ligase responsable des formes génétiques de la maladie de Parkinson (MP). En effet, plusieurs arguments soutiennent l'implication de la PK dans les gliomes.Des études ont montré que la PK présente une expression altérée dans les cas de cancers du sein et de la prostate. La PK possède également une fonction de facteur de transcription. Elle est capable de se fixer à l’ADN de p53 et inhibe sa transcription. p53 est un suppresseur de tumeur fréquemment inactivé (50% des cancers). Une étude a mis en évidence l'existence de mutations somatiques de la PK spécifiques des cancers cérébraux. Mon projet s'est articulé autour de trois axes. 1- PK et Maladie d’Alzheimer. Elle active la transcription de la préséniline 1 et d’inhiber celle de la préséniline 2. 2- PK et MP. La PK par l’intermédiaire de p53 régule XBP-1, un facteur de transcription notamment activé par le stress du réticulum, qui à son tour régule l’expression de DJ-1. 3- PK et gliomes. Nous avons observé une diminution d’expression de la PK corrélé à l’augmentation d’expression de p53 dans des biopsies de gliomes. Nous avons alors montré que p53 est capable d’activer la synthèse de la PK, effet aboli par des mutations de p53 dans les gliomes. / Gliomas are the most common form of brain tumor, the etiology of which remains unknown. Several epidemiological studies have shown the existence of a correlation between neurodegenerative diseases and brain tumor. We hypothesis that these two pathology share common molecular denominators. Here I study the role of parkin (PK) an ubiquitin ligase responsible of early onset Parkinson diseases. Several arguments support the involvement of PK in glioma. Studies have shown that PK expression is alterated in many types of cancers. PK is also a transcription factor which can bind to p53 DNA and inhibits its transcription. P53 is a tumor suppressor often find inactivate in cancers (50%). There is evidence of specific somatic mutations found in glioma. My work was organize according to three axes 1- PK and Alzheimer disease: PK activates préséniline 1 expression and inhibits préséniline 2. 2- PK through XBP-1 regulates p53, a transcription factor activated by reticulum stress, which in turn regulates the expression of DJ-1. 3- PK and Glioma: There is a decrease in parkin expression that can be correlated to p53 expression increase in glioma biopsies. I show that p53 is able to activate PK synthesis, a mechanism abolish by p53 mutations in tumors.

No description available.

<p>   </p> <p>Environmental toxicants such arsenic (As) and lead (Pb) are chemicals that enter the environment and can result in adverse health effects humans, especially during development. This dissertation work evaluated As and Pb to determine if developmental toxicity significantly changes at lethal and sub-lethal mixture concentrations using the zebrafish model. Joint action models were applied to survival data to determine the type of interaction. Metal exposures were from 1-120 hours post fertilization (hpf). As concentrations were 0-7,500 ppb. Pb concentrations were 0–100,000 ppb. The LC25, LC50, and LC75 values at 120 hpf from single metal exposures were used to select mixture concentrations for modeling. The survival data indicated an additive effect occurred at lethal concentrations. </p> <p>The impact of the mixture on behavior, morphology, and gene expression was then evaluated at sub-lethal concentrations of 10 and 100 ppb As and Pb individually or in mixtures. Data was analyzed with a repeated measures ANOVA (behavior) or an ANOVA (morphology and qPCR) with the least significant difference test (α=0.05). Zebrafish larvae exposed to 10 ppb As exhibited hyperactivity in all dark phases for the distance moved, time moving, and velocity, while those exposed to 10 ppb Pb only showed an increase in distance moved and velocity in the first dark phase. The 10 ppb mixture was found to have an intermediate impact with increased time moving in all dark phases and increased distance moved and velocity only in the first dark phase. In contrast, hyperactivity was observed only in the 100 ppb As and 100 ppb mixture treatment in the last two dark phases for time spent moving. No significant behavioral alterations occurred in the 100 ppb Pb treatment. A decrease in mean brain length and brain length ratio to the total length in the 10 ppb mixture was observed with no significant morphology changes observed for head length, head width, or total length. Alternatively, measurements of cerebral vasculature morphology in the mesencephalon (midbrain) and cerebellum (hindbrain) uncovered decreased total vascularization at 72 hpf (exposure 1-72 hpf) in both brain regions. This decrease occurred in all treatment groups in the mesencephalon and in all treatment groups, except the 100 ppb Pb and 10 ppb As treatment groups in the cerebellum. In addition, decreased sprouting and branching occurred in the mesencephalon, while only decreased branching was measured in the cerebellum. The 10 ppb Pb treatment group showed unique perturbations in several cerebral vasculature endpoints evaluated, which was also observed in a specific gene expression alteration pattern different from the other treatment groups. To identify molecular changes associated with these changes, expression of genes related to angiogenesis and vasculogenesis (i.e., <em>vegfaa, wnt7aa,</em> and<em> lrp1aa</em>) and genes associated with tight junctions (i.e., <em>cldn5a</em> and <em>cldn5b</em>) were assessed at 72 hpf. Increased <em>cldn5b</em> expression was detected in all treatment groups, while <em>cldn5a </em>was increased in only the 10 ppb Pb treatment group. In addition, <em>wnt7aa</em> was only decreased in the 10 ppb Pb treatment group. Alternatively, <em>vegfaa</em> was increased in the 100 ppb As and 100 ppb mixture treatment groups and no changes were detected for <em>lrp1aa</em>. In summary, cerebral vascular toxicity outcomes in the 10 ppb mixture treatment were primarily driven by changes in the 10 ppb Pb treatment group, while perturbations in the 100 ppb mixture treatment group aligned with the 100 ppb As alterations. In addition, the non-linear dose response for 10 and 100 ppb Pb treatment groups agree with observations in prior studies. qPCR results indicate that both metals together and separately alter cerebral vasculature development at environmental regulatory levels. </p> <p>Lastly, with the increase in the prevalence of neurodegenerative diseases increasing globally, there is a need to evaluate more therapeutics at a high through-put pace and to pinpoint the cause of the sporadic cases. CRISPR-Cas9 technology offers a relatively inexpensive, reliable, and precise advantages over its predecessors when it comes to producing mutant disease models of neurodegeneration. A method to create the expression vector needed for creation of a CRISPR Cas9 knock-in model is detailed in this dissertation. The methodology to insert a chimeric DNA sequence contain human DNA has been created, and the <em>in silico</em> assays used to produce the reactant for this methodology were successful. It has been determined that the efficiency of this knock-in method is limited to the success of producing the chimeric model which is limited itself by the number of molecules included into the chimeric sequence. Overall, the results show that the overlap primers designed in silico need to be re-designed to improve efficiency of the initial reactions required to produce the first plasmid containing the required machinery for a successful knock in of exogenous human DNA.</p>

Locomotion in vertebrates depends on proper formation and maintenance of neuronal networks in the hind-brain and spinal cord. Malformation or loss of factors required for proper maintenance of these networks can lead to severe neurodegenerative diseases limiting or preventing locomotion. A powerful tool to investigate the genetic and cellular requirements for development and/or maintenance of these networks is a collection of zebrafish mutants with defects in motility. The zebrafish mutant quetschkommode (que) harbors a previously unknown gene defect leading to abnormal locomotor behavior. Here I show that the que mutants display a seizure-like behavior starting around four days post fertilization (dpf) that is characterized by a lack of an initial high amplitude body bend (C-bend) and simultaneous contra-lateral contractions leading to a seizure-like phenotype and paralysis. Peripheral nerve recordings show a significant increase in the number of initiated swimming bouts and overlap between left and right motor neuron activity. These data suggest that the que mutation leads to defects in nervous system function, at the level of motor neurons or central control of motor neurons. I have genetically mapped the que locus to a 0.36cM interval on chromosome 22 using meiotic mapping. I identified a splice mutation in the gene `dihydrolipoamide branched-chain transacylase E2' (dbt) as defective in que mutants. An orthologous mutation in humans lead to Maple Syrup Urine Disease (MSUD), a devastating metabolic disorder leading to seizures, mental retardation, coma and neonatal death if untreated. In zebrafish, dbt is expressed throughout early development and dbt transcripts become enriched in the hind-brain as well as in the gut and liver by 96 hpf. In MSUD patients levels of branched chain amino acids (BCAA) and their keto acids are significantly increased due to the essential role of the dbt enzyme for the BCAA metabolic pathway. The que mutation causes a significant increase of branched chain amino acids in the zebrafish mutant and a strong decrease of neurotransmitters such as glutamate and GABA as well as precursors like glutamine. I hypothesize that reduced neurotransmitter levels in que lead to the observed motility phenotype. Consistent with this hypothesis, I show a tissue specific reduction of glutamate in the hind-brain and spinal cord of que mutants. To evaluate the que mutant's potential as a vertebrate model for MSUD I performed a pilot drug screen using a selection of metabolites of the pathway as well as diet additives currently evaluated in clinical trials. Conversely, application of phenylbutyrate, one of the diet additives, had a beneficial influence on swimming abilities of que mutant embryos, while the keto acid α-ketoisocaproate (KIC), one of the elevated keto acids in human patients, decreased the percentage of larvae capable of swimming. These results help establish the zebrafish que mutant as a new model for MSUD disease that can be used to further the understanding of this disorder and to help identify therapeutic agents.

In the field of gerontology, there is a question regarding whether certain diseases can accelerate the progression of other diseases. Given that there is no existing cure for dementia, there is an undeniable upsurge in demand for research concerning identifying preventive measures that influence the onset or development of cognitive decline. Cerebral atherosclerosis is characterized by the thickening of artery walls within the brain. Many studies have shifted their focus on examining the role nutrients and food play in preventing cognitive decline, dementia, cerebral atherosclerosis, and Alzheimer’s disease (AD). This thesis provides a comprehensive overview of existing observational and clinical trial evidence published up to date for the association of dietary intervention between cognitive health, dementia, AD, and cerebral atherosclerosis.

Editorial on the Research Topic. Obesogenic Environmental Conditions Affect Neurodevelopment and Neurodegeneration

Yes / Spinal muscular atrophy, the leading genetic cause of infant mortality, is a motor neuron disease caused by low levels of survival motor neuron (SMN) protein. SMN is a multifunctional protein that is implicated in numerous cytoplasmic and nuclear processes. Recently, increasing attention is being paid to the role of SMN in the maintenance of DNA integrity. DNA damage and genome instability have been linked to a range of neurodegenerative diseases. The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. Instability in rDNA has been associated with cancer, premature ageing syndromes, and a number of neurodegenerative disorders. Here, we report that SMN-deficient cells exhibit increased rDNA damage leading to impaired ribosomal RNA synthesis and translation. We also unravel an interaction between SMN and RNA polymerase I. Moreover, we uncover an spinal muscular atrophy motor neuron-specific deficiency of DDX21 protein, which is required for resolving R-loops in the nucleolus. Taken together, our findings suggest a new role of SMN in rDNA integrity.

According to the National Institute of Neurological Disorders and Stroke, there are more than 600 neurologic disorders that affect approximately 50 million Americans each year. The $91 billion dollars spent by Medicare on Alzheimer's disease and other dementias in 2005 is projected to increase to $189 billion by 2015 [4]. The existence of neural stem cells (NSC's) and neurogenesis makes neural regeneration a viable option. The ethical barriers of using embryonic stem cells, rejection of the transplanted cells, and possible tumor formation, are only a few of the problems that face stem cell transplantation, a widely considered option to repopulate the brain with cells. A noninvasive pharmaceutical approach that can promote neuron regeneration and recovery would be the key to curing many neurodegenerative diseases. The development of MS-818 as a non-invasive enhancer of the proliferation process of NS Cs is revolutionary for the treatment of neurodegenerative diseases. Due to the fact that its mechanism of action remains unknown, the full pharmacological potential of MS- 818 has not been fully exploited [8]. Isolating protein targets of MS-818 is key in identifying the molecular pathways responsible for its mechanism of action. UV-Vis analysis of MS-818 showed absorbance at 275-nm, and this data was used to calculate coupling yield. MS-818 coupled to the NHS-activated sepharose beads of the affinity column with 83% efficiency. Proteins were isolated from human embryonic kidney cells (HEK 293 cells) and applied to the column. Bradford assay confirmed that bound proteins eluted in a concentration dependent manner when an MS-818 gradient was applied to the column. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the eluate revealed two sets of polypeptides migrating at 37-75 kDa and 100-150 kDa. In addition, some trace polypeptides in the sub-35 kDa region could be seen. Although we have not yet identified specific proteins that MS-818 interacts with, we were able to successfully to isolate such proteins.

The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA), a non-protein amino acid, first attracted attention in correlation to reports of high incidence of the unusual neurological disease amyotrophic lateral sclerosis/Parkinsonism-dementia (ALS/PDC) among the people of Guam in the South Pacific Ocean. Experimental studies have revealed that BMAA causes neuronal cell death. The neurotoxin is suggested to act via excitotoxicity through interaction with glutamatergic receptors. More importantly, BMAA is suggested to misincorporate in the synthesis of proteins, and contribute to protein misfolding and/or deleterious aggregation, which are hallmarks of several neurodegenerative disorders. A selective uptake of BMAA in the rat neonatal hippocampus can interfere with brain development, causing learning and memory impairments in adult rats. The aim of the present study was to investigate the effects of BMAA in human neuroblastoma SH-SY5Y cells. These cells were exposed to BMAA (10 μM, 50 μM, 100 μM or 500 μM) for 72 hours, and the expression of five selected proteins, including heat shock protein-27 (HSP-27), lysosomal associated membrane protein-1 (LAMP-1), CCAAT-enhancer-binding protein homologous protein (CHOP), Golgi associated plant pathogenesis related protein-2 (GLIPR-2), and glucose regulated protein-78 (GRP-78). They were carried out with immunohistochemistry (IHC). Results revealed an increased expression of all selected proteins, which indicates an uptake and shows the effects of BMAA in the cell cultures. Taken together, BMAA caused cellular stress, including endoplasmic reticulum (ER) stress that is correlated with HSP-27, LAMP-1, CHOP, GLIPR-2, and GRP-78. Further studies are needed in order to support the results. The experiments require being repeated using the same biomarkers as well as a combination of them with other biomarkers to elucidate the effects of BMAA.