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Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN
Contributor:Konopka, Witold : Supervisor ; Wojda, Urszula : Supervisor
Publisher:Instytut Biologii Doświadczalnej im. M. Nenckiego PAN
Place of publishing: Date issued/created: Description:201 pages : illustrations ; 30 cm ; Bibliography ; Summary in English
Degree name: Degree discipline : Degree grantor:Nencki Institute of Experimental Biology PAS ; degree obtained: 29.06.2022
Type of object: Subject and Keywords:AgRP/NPY neurons ; CRISPR/Cas9 ; Dicer ; microRNA ; Obesity ; Metabolism
Abstract:
Food is the foundation of the survival pyramid while hunger is the primary drive that motivates the search for and acquisition of nourishment. The brain is the locus of the superior centers involved in the regulation of hunger and satiety. The arcuate nucleus, located in the immediate vicinity of the median eminence in the hypothalamus, is the primary first-order center processing information about the body's energy status. Its composition includes two populations of opposing neurons: AgRP/NPY - stimulating food intake and POMC/CART - responsible for promoting satiety and appetite suppression. Any disturbances within this center and in the communication of the arcuate nucleus with second-order neurons in other centers may lead to changes in eating behavior and the development of metabolic diseases such as obesity. This dissertation chiefly aims to investigate the involvement of AgRP/NPY neurons in the development of obesity in animals with the neurospecific deletion of the Dicer1 gene. Dicer is an enzyme the endonucleolytic action of which leads to the production of mature forms of microRNA (miRNA) molecules regulating the translation process. Cells deprived of the Dicer1 gene lack functional, canonical microRNAs. The research problem required the use of several transgenic mice models. All models were based on the Cre-loxP system, where recombination was induced by Tamoxifen in some and administration of the AAV viral vector regulating the inserted transgene with the AgRP promoter or by using CRISPR/Cas9 technology in others. Analysis and modification of successive transgenic models resulted in high specificity of the introduced change, selectively in AgRP/NPY neurons. Analysis of the obtained research models evidenced that the system with the induced Dicer1 deletion in AgRP neurons (AgRPCreERT2Dicerfl/fl) does not allow for conclusions about the involvement of these neurons, due to the insufficient level of recombination in this lineage. The remaining models provided significant information. The model with the inducible loss of microRNA in mature CaMKIIα neurons and the simultaneous loss of both Npy alleles (NPY-KO/DicerCKO) excluded the involvement of NPY as a key stimulant of food intake. Meanwhile, experiments on Dicerfl/fl mice subjected to intracerebral injections of the AAV- AgRPCre vector into the arcuate nucleus, showed the significant involvement of AgRP neurons in the development of microRNA-dependent obesity. Moreover, they revealed a quantitative relationship between the number of AAV-vector particles introduced determining the number of modified neurons and the increased nutritional requirements leading to increased body weight. Mice expressing Cre and Cas9 in AgRP neurons (AgRPCreCas9) proved to be the model generating the highest degree of specificity in the targeted modification of Dicer1 in AgRP neurons. These animals, following intracerebral administration of the AAV-guide2Dicer vector, developed massive obesity associated with severe appetite while also revealing a sex- dependent effect, where only females lacking microRNA in AgRP neurons showed a significant increase in appetite and body weight. The above observations imply the important role of microRNA in regulating the functions of AgRP/NPY neurons. Selective loss of microRNA in hunger neurons leads to impairment of their functioning, which is manifested by increased appetite and development of obesity. The number of modified hunger neurons determines the magnitude of the effect, indicating there is a subtle balance between the signals of hunger and satiety. Sexual differences in the observed phenotype suggest microRNA plays a role in modulating hormone-dependent pathways.
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Digitizing institution:Nencki Institute of Experimental Biology of the Polish Academy of Sciences
Original in:Library of the Nencki Institute of Experimental Biology PAS
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