In this thesis the natural cellular trafficking has been used to target the expression of intracellular antibodies to specific subcellular compartments. Recombinant antibodies in the fonn of single chain Fab variable fragments (scFvs) were expressed in fusion with GFP and targeted into intracellular organelles of neurqnal cells. It was first verified that neurons can correctly target intracellularly expressed scFvs to the nucleus, secretory pathway and endoplasmic reticulum (ER) when fused with an appropriate, nuclear localization sequence (NLS), secretory leader (sec), or KDEL ER retention signal (KDEL). Since neurotrophin receptors are located in the distal dendrites and are the focus of my study, specific mRNA targeting elements were used to reach particular cell compartments such as distal dendrites in neurons. Three different RNA sequences were compared in order to detemline the most advantageous conditions for neuronal expression: the hnRNP A2 Responsive Element (A2RE) for transport of Myelin Basic Protein (MBP) mRNA into oligodendrocyte processes, the 3 'UTR of Ca2 + - calmodulin-dependent protein kinase II a (CaMKila) that regulates CaMKIIa mRNA trafficking, and the first 94 nucleotides of the Ca.MKIJa UTR. The RNA targeting experiments described in this thesis show that it is possible to target an scFv mRNA to dendrites of transfected neurons, and indicates that the A2RE as the most powerful element among the studied sequences. Having validated the protein and RNA targeting of scFvs, these technologies were exploited to interfere with the NGF receptors, TrkA and p75NTR. Three different scFvs were selected to reach this goal: a TrkA MNAC as a neutralizing antibody, a TrkA, and a p75 scFvs as intracellular anchors. After an initial characterization of the three scFvs, the efficiency of the KDEL retained scFvs as intracellular anchors was demonstrated in stably transfected C6 cells. The consequences of reducing the number of NGF receptors present on the cell surface was evaluated in PC12 and primary hippocampal neurons after transient transfection. Tue inhibition of differentiation in PC 12 cells, and the inhibition of signal transduction both in PC12 cells and neurons, demonstrate that ER-retained scFvs are able to interfere effectively with neurotrophins induced signal transduction, confirming that phenotypic knock-out occurred. With the .same type of experiments it was also possib.le to observe, with. a completely novel approac:h, a complex formation between p75NTR and Trk receptors. Fillally, the new tools developed were used as a basis for the design of intrabody constructs to create new transgenic mice for the study of Alzheimer's disease.
Use of intracellular antibodies to study the cellular biology of neuronal cells / Pastore, Giada. - (2003 Nov 24).
Use of intracellular antibodies to study the cellular biology of neuronal cells
Pastore, Giada
2003-11-24
Abstract
In this thesis the natural cellular trafficking has been used to target the expression of intracellular antibodies to specific subcellular compartments. Recombinant antibodies in the fonn of single chain Fab variable fragments (scFvs) were expressed in fusion with GFP and targeted into intracellular organelles of neurqnal cells. It was first verified that neurons can correctly target intracellularly expressed scFvs to the nucleus, secretory pathway and endoplasmic reticulum (ER) when fused with an appropriate, nuclear localization sequence (NLS), secretory leader (sec), or KDEL ER retention signal (KDEL). Since neurotrophin receptors are located in the distal dendrites and are the focus of my study, specific mRNA targeting elements were used to reach particular cell compartments such as distal dendrites in neurons. Three different RNA sequences were compared in order to detemline the most advantageous conditions for neuronal expression: the hnRNP A2 Responsive Element (A2RE) for transport of Myelin Basic Protein (MBP) mRNA into oligodendrocyte processes, the 3 'UTR of Ca2 + - calmodulin-dependent protein kinase II a (CaMKila) that regulates CaMKIIa mRNA trafficking, and the first 94 nucleotides of the Ca.MKIJa UTR. The RNA targeting experiments described in this thesis show that it is possible to target an scFv mRNA to dendrites of transfected neurons, and indicates that the A2RE as the most powerful element among the studied sequences. Having validated the protein and RNA targeting of scFvs, these technologies were exploited to interfere with the NGF receptors, TrkA and p75NTR. Three different scFvs were selected to reach this goal: a TrkA MNAC as a neutralizing antibody, a TrkA, and a p75 scFvs as intracellular anchors. After an initial characterization of the three scFvs, the efficiency of the KDEL retained scFvs as intracellular anchors was demonstrated in stably transfected C6 cells. The consequences of reducing the number of NGF receptors present on the cell surface was evaluated in PC12 and primary hippocampal neurons after transient transfection. Tue inhibition of differentiation in PC 12 cells, and the inhibition of signal transduction both in PC12 cells and neurons, demonstrate that ER-retained scFvs are able to interfere effectively with neurotrophins induced signal transduction, confirming that phenotypic knock-out occurred. With the .same type of experiments it was also possib.le to observe, with. a completely novel approac:h, a complex formation between p75NTR and Trk receptors. Fillally, the new tools developed were used as a basis for the design of intrabody constructs to create new transgenic mice for the study of Alzheimer's disease.File | Dimensione | Formato | |
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