3 Results
3.1 Characterisation of the used Drosophila transgenes
3 Results
As described in chapter 1.9 the aim of this work is to identify modifiers of Tau induced pathology. This was done in a genetic modifier screen using an RNAi-‐based approach and the REP induced by Tau as a readout (see chapter 1.8.1). Thus, characterisations of the used transgenes were performed according to their REP induction and life span.
3.1 Characterisation of the used Drosophila transgenes
Two transgenic lines modelling Tau-‐induced toxicity were kindly provided by Mel Feany [125]. The two transgenes Tau[WT] (P{w[+mC]=UAS-hTau[WT]}) and Tau[R406W]
(P{w[+mC]=UAS-hTau[R406W]}) were used to model Tau-‐induced neurodegeneration (FTDP-‐17). Besides Tau, two other transgenes were evaluated for induction of a REP, when expressed in the compound eye: Aβ42 (P{[+mC]=UAS-Aβ42}) used to model amyloid-‐
induced AD [241] and Q78 (P{[+mC]=UAS-MJD.tr-Q78}) used to model the poly-‐glutamine disease spinocerebellar ataxia type 3 [248]. These two Tau unrelated transgenes were utilised to analyse specificity of found modifiers for Tau pathology.
3.1.1 Rough eye phenotypes of the models
For the evaluation of transgene-‐induced toxicity in photoreceptors, the transgenes were expressed in post-‐mitotic cells of the compound eye (GMR-Gal4 [233]). Several controls for eye appearance were used (Figure 9 A-‐D). In contrast to the wildtype eye phenotype (Figure 9 A), a very subtle REP was visible after GMR mediated Gal4 expression (Figure 9 B). Comparable phenotypes could be observed in GFP expression (transgene P{[+mC]=UAS-eGFP}4) and transcription of a control shRNA (shRNA directed against ZIC4) (Figure 9 C, D).
GMR-‐Gal4 mediated expression of Tau[WT] and Tau[R406W] showed a severe REP, with loss of texture and reduced size of the compound eye (Figure 9 E, F). In flies expressing Tau[R406W], necrotic spots in the eye could be observed occasionally. Since the Tau[R406W]-‐induced phenotype was found to be more robust, with only minor inter-‐
individual differences compared to Tau[WT], GMR-‐mediated expression of the mutant variant Tau[R406W] was chosen for further analysis and the modifier screen.
The REP induced by expression of Aβ42 (Figure 9 G) did not differ from the control phenotypes (Figure 9 B-‐D) (in contrast to published data [241]). Nevertheless,
enhancement of the phenotype induced by modifiers should be observable. Expression of Q78 (a C-‐terminal fragment of MJD containing a stretch of 78 glutamines) induced a severe REP. Although the eye size is not affected by Q78 expression, texture and ommatidial structure are impaired and dints as well as necrotic spots occur (Figure 9 H).
3.1.2 Developmental effects of Tau expression
As the REP is a sign of neurodegeneration already present after hatching of the adult fly, larval eye progenitors were examined to determine the onset of neurodegenerative effects. The structure in Drosophila L3 larvae, which will form the compound eye, is called eye imaginal disc. The eye imaginal disc is a two-‐layered epithelial sack-‐like structure connected posterior to the optic lobes and anterior to the antenna imaginal disc (Figure 10 A). During development specification of neurons takes place in a spatiotemporal order starting posterior. The so called morphogenic furrow (MF) represents the border of neuron specification dividing post-‐mitotic neuronal precursor cells on the posterior and
Figure 9: Phenotypes induced by GMR-mediated expression of the different transgenes.
Compound eye phenotypes of wildtype flies (A), control situations (B-‐D) and flies expressing transgenes modelling neurodegenerative diseases (E-‐H). Compared to wildtype flies (A), the GMR-Gal4 driver alone induces a subtle REP (B), which is also present in GMR-Gal4 mediated GFP expression (C) and control shRNA transcription (D). Expression of either Tau[WT] (E) or Tau[R406W] (F) induces a severe REP with disturbances of ommatidial structure and eye size. The REP induced by Gal4-‐mediated expression of Aβ42 (G) is not distinguishable from the phenotypes of control situations. Severe changes of eye texture, accompanied by dints and necrotic spots are found in expression of the Q78 fragment (H). Orientation of the images is dorsal-‐up and cranial-‐left. The magnification is depicted in D (bar = 200µm).
yet unspecified cells on the anterior side is [249, 250]. The GMR-‐mediated expression of transgenes is specific for post-‐mitotic cells posterior of the MF (Figure 10 B). Loss of cells leading to the REP observed in adult flies might be already visible in the imaginal disc of the Drosophila compound eye [229, 251]. To examine cell death events induced by Tau expression, eye disc of Drosophila were stained with acridine orange (AO). AO labels dying cells in situ, due to their inability to exclude AO. In these cells AO intercalates into DNA resulting in green fluorescence signal [252].
Eye imaginal discs of GMR-Gal4 flies, showed a small number of AO positive cells directly at the morphogenic furrow where supernumerary cells are eliminated (Figure 10 C). In eye imaginal discs with GMR-Gal4-‐induced Tau[R406W] expression an increased number of AO positive cells was detectable in the area posterior of the morphogenic furrow (Figure 10 D), suggesting that toxic effects of Tau[R406W] already induce cell death in the developing eye of Drosophila and are not exclusively mediated by long-‐term accumulation of a toxic protein species.
Figure 10: Developmental effects of Tau[R406W] expression in the eye imaginal disc of Drosophila.
Schematic view of the antenna imaginal disc (AID) and the eye imaginal disc (EID) (A). The morphogenic furrow (MF) is the border of cell differentiation starting posterior (post.). The differentiation results in post-‐
mitotic neuronal precursor cells (N) posterior of the MF. These resemble the area of GMR-Gal4-‐mediated expression (grey), also visible in GMR-‐driven GFP expression (B). In GMR-Gal4 flies AO-‐positive cells undergoing apoptosis are found directly at the MF (C). In eye imaginal discs with GMR-‐mediated expression of Tau[R406W] an increased number of AO-‐positive cells are found which are not restricted to the MF (D).
Orientation of the images is anterior-‐left and dorsal-‐up. Magnification is depicted in A (bar = 150 µm). B is an overlay of brightfield and fluorescence microscopy.
3.1.3 Comparison of Tau expression levels
To evaluate the expression levels in the screening stock, quantitative PCR (qPCR) analysis was performed. Transcript levels of endogenous Drosophila Tau (dTau) as well exogenous transgene Tau[R406W] (human Tau hTau) were quantified in RNA extracts from heads of flies expressing Tau[R406W] under control of the pan-‐neural elavC155-Gal4 driver. As a control, the driver line was used without any transgene expression. ΔΔCt analysis revealed no significant change of endogenous dTau mRNA levels in Tau[R406W]
expressing flies, compared to control flies, normalised to ACTG1 (Actin 5C) (Figure 11 A).
To evaluate Tau[R406W] transcript levels ΔCt analysis was performed comparing Tau[R406W] mRNA to dTau mRNA. Transcript levels of transgenic Tau[R406W] were significantly increased (1.8-‐fold, p=0.0006) compared to measured dTau mRNA levels (Figure 11 B).
Figure 11: Tau mRNA levels in the used Tau[R406W] model.
(A) The effect of elav-‐driven Tau[R406W] expression on endogenous dTau mRNA levels in total RNA preparations from the head. No significant change is measurable. (B) Comparison of dTau and Tau[R406W]
mRNA levels in total RNA preparations from head in flies with elav-‐driven Tau[R406W] expression. The Tau[R406W] transgene has 1.8 times higher transcription levels compared to endogenous dTau (standard t-‐
test: *** p<0.001).
3.1.4 Longevity of the disease models
Human neurodegenerative diseases are progressive disorders with ongoing loss of neuronal function, leading to reduced life span. This can also be modelled in Drosophila [125, 241, 248]. Therefore, transgenes are expressed via pan-‐neural elavC155-Gal4 driver.
Compared to GFP expression as a control, both Tau[R406W] and Aβ42 expression led to a significant decrease in life span (Figure 12 A). Pan-‐neural expression of Q78 under control of elavC155-Gal4 was already lethal during pupal stage and therefore could not be used for longevity analysis.