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.
