Dissertation zu Expressionsmustern von muskarinergen Acetylcholin-Rezeptoren des Subtypen 1,2,3 und 4 im Gehirn von DYT1 knock-in Mäusen.

DYT1 dystonia is an early onset central nervous system-based movement disorder characterized by uncontrolled sustained muscle contractions that can lead to debilitating abnormal postures. Though a genetic mutation in the gene TOR1A is responsible for most DYT1 cases, the low penetrance of the disease implicates additional genetic and environmental modifiers. Current therapeutic options for DYT1 dystonia are limited to symptomatic treatments with variable effectiveness. Currently, the underlying pathogenesis of this disease and the role of torsinA (torA), the protein product of TOR1A, in the development of this disease have yet to be established. In the first part of this thesis we aimed to further understand the effects of the TOR1A mutation at the molecular, cellular and organismal level in order to identify disease associated biomarkers that can be later used to measure the effectiveness of novel therapies. We found that expression of mutant torsinA (torA(ÄE)) in a cellular and an animal model of DYT1 had no significant effect on global transcription, despite its interaction with nuclear envelope proteins. Recent research has unearthed a role for microRNAs (miRNAs) in neuronal development and maturation. Consequently we explored whether torA(ÄE) expression in murine neural tissue was associated with changes in miRNA expression in young DYT1 knockin (KI) mice. Since the primary sight of dysfunction is still being debated, we profiled miRNA expression of the two strongest candidates, the striatum and cerebellum, both of which have well established roles in the control and coordination of muscle movements. We have identified several microRNAs that were uniquely altered in either the striatum or cerebellum and further research will be conducted to determine their usability as disease biomarkers. Finally, we were unable to identify motor phenotypes in either a DYT1 (KI) mice or a novel DYT1 transgenic model in open field, rotarod or staircase forepaw reaching tests. In the second part of this thesis we aimed to develop and evaluate the safety and efficacy of viral therapeutic RNAi constructs for in DYT1 murine models. DYT1 is an ideal candidate for this form of therapy due to its dominant inheritance, common mutation and potentially reversible phenotype. Virally delivered short-hairpin RNAs (shRNA) designed to knockdown torA(ÄE) in either an allele-specific or nonallele-specific manner were injected into the striatum of DYT1 transgenic or KI mice respectively. Unexpectedly, we found widespread lethal toxicity and behavioral abnormalities in mice injected with either therapeutic or control shRNAs that weren't observed in mice injected with no shRNAs. Further studies found that regions where toxic shRNAs were expressed corresponded with neuronal loss and glial activation. Finally, we found evidence that the severity of toxicity was influenced in part by the genetic background of the mice. In summary, the studies completed in this thesis contribute important information to the fields of dystonia pathogenesis and therapeutics, and more broadly pertain to the development of therapeutic gene silencing for neurological disease.

DYT1 dystonia (DYT1) is a disabling inherited neurological disorder with juvenile onset. The genetic mutation in DYT1 leads to the deletion of a glutamic acid (E) residue in the protein torsinA. The function of torsinA and how the mutation leads to DYT1 is poorly understood. We hypothesize that how efficiently the disease-linked mutant protein is cleared may be critical for DYT1 pathogenesis. Therefore we explored mechanisms of torsinA catabolism, employing biochemical, cellular, and animal-based approaches. We asked if torsinA(wt) and torsinA(DE) are degraded preferentially through different catabolic mechanisms, specifically the ubiquitin proteasome pathway (UPP) and autophagy. We determined that torsinA(wt) is cleared by autophagy while torsinA(DE) is efficiently degraded by the UPP suggesting degradation processes can modulate torsinA(DE) levels. Proteins implicated in recognizing motifs on torsinA(DE) for targeting to the UPP represent candidate proteins that may modify DYT1 pathogenesis. We examined how removal of the hydrophobic domain and mutation of glycosylated asparagine residues on torsinA altered stability and catabolic mechanism. We found the glycosylation sites on torsinA are important for stability modulate its degradation through the UPP. F-box G-domain protein 1 (FBG1) has been implicated in degradation of glycosylated ER proteins. We hypothesized that FBG1 would promote torsinA degradation and demonstrated that FBG1 modulates levels of torsinA in a non-canonical manner through the UPP and autophagy. We examined if lack of FBG1 in a torsinA(DE) mouse model altered motor phenotypes. We saw no effect which suggests FBG1 does not alter DYT1 pathogenesis despite its promotion of torsinA(DE) degradation. In addition, we explored a potential mechanism for the previously described role of torsinA in modulating cytoplasmic protein aggregation. We hypothesized this endoplasmic reticulum (ER) resident protein would indirectly alter cytoplasmic protein aggregation through modulation of ER stress. We employed a poly-glutamine expanded repeat protein and pharmacological ER stressors to determine that torsinA does not alter poly-glutamine protein aggregation nor ER stress in a mammalian system. In summary, this thesis suggests proteins involved in the catabolism of torsinA(DE) may modify DYT1 pathogenesis and that torsinA and its DYT1-linked mutant are model proteins for investigating ER protein degradation by the UPP and autophagy.

Στην παρούσα διατριβή μελετήθηκε το ντοπαμινεργικό και γλουταμινεργικό σύστημα των βασικών γαγγλίων, χρησιμοποιώντας δύο διαφορετικά μοντέλα ζώων. Ο πρώτος στόχος ήταν να εξεταστεί εάν η μεταφορά του γονιδίου TrkA σε νευρώνες της μέλαινας ουσίας (SN) ενήλικων επίμυων επιδρά στις νευροχημικές τους ιδιότητες, απουσία ή παρουσία εξωγενούς νευροαυξητικού παράγοντα (NGF) στο ραβδωτό σώμα. Η εκτοπική έκφραση του TrkA στην SN οδήγησε σε σημαντική μείωση του mRNA της υδροξυλάσης της τυροσίνης (TH), της TH ανοσοδραστικότητας και του mRNA του DAT στη δεξιά SN σε σύγκριση με την ετερόπλευρη, ενώ δεν βρέθηκε καμία διαφορά στο mRNA των υποδοχέων ντοπαμίνης D2 και της ειδικής δέσμευσης του [3Η]raclopride στην SN. Δεν παρατηρήθηκαν μεταβολές στις θέσεις δέσμευσης του [3Η]WIN35428 και της ανοσοδραστικότητας του DAT στο ομόπλευρο ραβδωτό σώμα, καθώς και στις θέσεις δέσμευσης των μετασυναπτικών υποδοχέων ντοπαμίνης D1 και D2, όπως καθορίζεται από τους ιχνηθέτες [3H]SCH23390 και [3Η] raclopride, αντίστοιχα. Επιπλέον, δεν βρέθηκαν σημαντικές μεταβολές στους υποδοχείς NMDA και AMPA. Τα αποτελέσματα αυτά δείχνουν ότι η εκτοπική έκφραση του TrkA στην SN ρυθμίζει αρνητικά την ΤΗ και οδηγεί σε ανεξάρτητες από τον NGF αποκρίσεις. O δεύτερος στόχος της διατριβής ήταν η μελέτη του ντοπαμινεργικού συστήματος σε ένα μοντέλο DYT1 δυστονίας μυός. Σε διαγονιδιακούς μυς με υπερκινητική και μη υπερκινητική συμπεριφορά παρατηρήθηκε μείωση των υποδοχέων ντοπαμίνης D2 στο ραβδωτό σώμα, όπως προσδιορίστηκε από τη δέσμευση του [3H]raclopride, και τoυ mRNA των D2 στην SNpc, σε σχέση με μη διαγονιδιακούς μυς. Δεν παρατηρήθηκε διαφορά στη δέσμευση του [3H]SCH23390 ή του [3H]WIN35428 στο ραβδωτό σώμα διαγονιδιακών μυών. Τα δεδομένα προτείνουν μία πιθανή εμπλοκή της ντοπαμινεργικής νευροδιαβίβασης στην παθοφυσιολογία της DY1 δυστονίας. / In the present thesis we examined the dopaminergic and glutamatergic neurotransmission systems of basal ganglia, using two different animal models. The first goal was to investigate whether TrkA gene transfer into substantia nigra (SN) neurons of adult rats influence some of their neurochemical properties, in the absence or presence of exogenous nerve growth factor (NGF) delivery in the striatum. Ectopic expression of TrkA in SN resulted in a significant decrease of tyrosine hydroxylase (TH) immunoreactivity, TH mRNA and DAT mRNA expression in the right SN compared to the contralateral side, while no difference was found in the mRNA expression of D2 DA receptors and [3H]raclopride binding in SN. No significant changes were seen in the density of DAT by measuring [3H]WIN35428 binding sites and DAT immunoreactivity in the ipsilateral striatum, as well as in the number of postsynaptic striatal D1 and D2 receptor binding sites, as determined by [3H]SCH23390 and [3H]raclopride, respectively. Furthermore, no significant changes were found in NMDA and AMPA receptors. These data suggest that ectopic TrkA expression in SN downregulates TH in nigral dopaminergic neurons and elicits NGF-independent responses. The second goal of the present thesis was to examine the dopaminergic system of basal ganglia in a mouse model of DYT1 dystonia. A decrease in striatal D2 binding sites, measured by [3H]raclopride binding, and D2 mRNA expression in substantia nigra pars compacta (SNpc) was revealed in affected and unaffected transgenic mice when compared with non-transgenic. No difference in D1 receptor binding and DAT binding, measured by [3H]SCH23390 and [3H]WIN35428 binding, respectively, was found in striatum of transgenic animals. These data suggest a possible involvement of dopamine neurotransmission in the pathophysiology of DYT1 dystonia.