Neurodegenerative diseases are a diverse group of progressive diseases that include Alzheimer’s disease (AD) and prion disease. Although these diseases differ in etiology, they share a number of similarities at the molecular level. For instance, microRNA (miRNA), small RNA molecules that post-translationally regulate gene expression, are often differentially regulated during disease. A previous study identified key miRNA that are dysregulated during prion disease in the hippocampus. Of these miRNA, miR-16-5p is of particular interest, as it has also been found to be dysregulated in AD. The objective of this thesis is to characterize the role of miR-16-5p within hippocampal neurons in order to understand its function during neurodegeneration. It is hypothesized that hippocampal miR-16-5p, given its induction in hippocampal neurons during preclinical disease, plays a role in regulating the dendritic remodeling and synaptic pruning that is the earliest pathological feature of neuronal degeneration in prion disease. To address this hypothesis, primary hippocampal neurons were dissected from embryonic day 18 mice and treated with a lentiviral vector at maturity. This vector either encoded miR-16 or miRZIP-16, causing overexpression or knockdown of miR-16, respectively. Immunoprecipitation of the miRNA-16 enriched RISC complex was then performed, and the co-immunoprecipitated target mRNA was subjected to a whole genome microarray. Analysis of microarray data in Ingenuity Pathway Analysis pinpointed 181 genes involved in neuronal morphology and neurological disease targeted by miR-16. In particular, the MAPK/ERK pathway was targeted at TrkB, MEK1 and c-Raf. This is of interest, as we know that this pathway is disrupted in other neurodegenerative diseases and is directly implicated in neuronal morphology. Subsequent morphological analysis revealed that overexpression of miR-16 in neuronal cells decreased neurite length and branching, consistent with the downregulation of components of the MAPK/ERK pathway. In conclusion, miR-16 targets many mRNA transcripts within the hippocampus that are important members of pathways involved in neuronal development and neurodegeneration, including the MAPK/ERK pathway. / February 2017

Les microARNs sont des petits ARNs non codants d'environ 22 nucléotides qui régulent négativement la traduction de l'ARN messager cible (ARNm) et ont donc des fonctions cellulaires. Le microARN-16 (miR-16) est connu pour ses effets antiprolifératifs. Nous avons observé que l’expression de miR-16 est diminuée dans les cellules endothéliales humaines sénescentes et quiescentes en comparaison à des cellules prolifératives. Une analyse informatique des sites potentiels de liaison de miR-16 prévoit que GLUT-4, un transporteur du glucose insulinodépendant, pourrait être une cible potentielle du miR-16. Nous avons donc testé l'hypothèse que miR-16 régule négativement le métabolisme du glucose cellulaire. Dans des HUVEC, l'inhibition de miR-16 endogène avec des anti-miRNA oligonucléotides (AMO) augmente les niveaux protéiques de GLUT-4 de 1,7 ± 0,4 fois (p=0,0037 ; n=9). Dans des souris nourries avec un régime alimentaire normal ou riche en graisse et en sucre, l’expression de GLUT-4 dans le muscle squelettique a tendance à corréler négativement avec les niveaux de miR-16 (p=0,0998, r2=0,3866, n=4). Ces résultats suggèrent que miR-16 est un régulateur négatif de GLUT-4 et qu’il pourrait être impliqué dans la régulation du métabolisme cellulaire du glucose. / MicroRNAs are small noncoding RNAs of approximately 22 nucleotides that negatively regulate translation of the target messenger RNA (mRNA) and therefore have cellular functions. MicroRNA-16 (miR-16) is known to display anti-proliferative effects. We observed that miR-16 was down-regulated in non-proliferative human senescent endothelial cells. Computational analysis of the potential binding sites of miR-16 predicted that GLUT-4, an insulin-dependent glucose transporter, is a potential target of miR- 16. We therefore tested the hypothesis that miR-16 down-regulates cellular glucose metabolism. In HUVEC, inhibition of using anti-miRNA oligonucleotides (AMO) endogenous miR-16 up-regulated GLUT-4 protein levels 1,7 ± 0,39 folds (p=0,0037; n=9). In mice fed a regular or high fat diet, skeletal muscle expression of GLUT-4 tended to negatively correlate with miR- 16 levels (p=0,0998, r2=0,3866, n=4). These results suggest that miR-16 is a negative regulator of GLUT-4 and may be involved in the regulation of cellular glucose metabolism.

Les microARNs sont des petits ARNs non codants d'environ 22 nucléotides qui régulent négativement la traduction de l'ARN messager cible (ARNm) et ont donc des fonctions cellulaires. Le microARN-16 (miR-16) est connu pour ses effets antiprolifératifs. Nous avons observé que l’expression de miR-16 est diminuée dans les cellules endothéliales humaines sénescentes et quiescentes en comparaison à des cellules prolifératives. Une analyse informatique des sites potentiels de liaison de miR-16 prévoit que GLUT-4, un transporteur du glucose insulinodépendant, pourrait être une cible potentielle du miR-16. Nous avons donc testé l'hypothèse que miR-16 régule négativement le métabolisme du glucose cellulaire. Dans des HUVEC, l'inhibition de miR-16 endogène avec des anti-miRNA oligonucléotides (AMO) augmente les niveaux protéiques de GLUT-4 de 1,7 ± 0,4 fois (p=0,0037 ; n=9). Dans des souris nourries avec un régime alimentaire normal ou riche en graisse et en sucre, l’expression de GLUT-4 dans le muscle squelettique a tendance à corréler négativement avec les niveaux de miR-16 (p=0,0998, r2=0,3866, n=4). Ces résultats suggèrent que miR-16 est un régulateur négatif de GLUT-4 et qu’il pourrait être impliqué dans la régulation du métabolisme cellulaire du glucose. / MicroRNAs are small noncoding RNAs of approximately 22 nucleotides that negatively regulate translation of the target messenger RNA (mRNA) and therefore have cellular functions. MicroRNA-16 (miR-16) is known to display anti-proliferative effects. We observed that miR-16 was down-regulated in non-proliferative human senescent endothelial cells. Computational analysis of the potential binding sites of miR-16 predicted that GLUT-4, an insulin-dependent glucose transporter, is a potential target of miR- 16. We therefore tested the hypothesis that miR-16 down-regulates cellular glucose metabolism. In HUVEC, inhibition of using anti-miRNA oligonucleotides (AMO) endogenous miR-16 up-regulated GLUT-4 protein levels 1,7 ± 0,39 folds (p=0,0037; n=9). In mice fed a regular or high fat diet, skeletal muscle expression of GLUT-4 tended to negatively correlate with miR- 16 levels (p=0,0998, r2=0,3866, n=4). These results suggest that miR-16 is a negative regulator of GLUT-4 and may be involved in the regulation of cellular glucose metabolism.