All experiments so far have been conducted in fly polyQ models with shRNA targeting the expression of the fly orthologue CG3808 of the human TRMT2A gene.
Unfortunately, there are no classical loss-of-function alleles of CG3808 available. In addition, the lack of independent RNAi lines to silence CG3808 prevented us from confirming our findings. Although Drosophila proved to be a feasible and beneficial tool for analysis of polyQ modifier genes, it is of crucial importance to confer the insights gained regarding amelioration of aggregation and toxicity to a mammalian system. Moreover, reconfirmation of the beneficial effects of TRMT2A silencing in the context of polyQ-induced toxicity in a vertebrate system would be desirable. Demonstrating the favourable activity of TRMT2A silencing in polyQ diseases would eventually deduce a universal mechanism conserved between flies and vertebrates, highlighting the experimental rational of our screen.
5.6.1 Generation of stable TRMT2A knockdown HEK cells
For the cell culture experiments human embryonic kidney cells (HEK293) were utilised. The high transfection efficiency and general robustness regarding both growth and protein production rendered HEK cells a feasible model system for the polyQ investigations.
For stable silencing of TRMT2A expression, five different shRNA lentiviral transduction particles, targeting individual human TRMT2A mRNA sequences, were purchased for treatment of HEK293 cells. Additionally, one non-target shRNA control viral strain was used, coding for an shRNA without any known cellular targets. Subsequent to viral transduction (at Department of Biochemistry, University Medical Centre Aachen) with different multiplicities of infection (MOI), cell colonies having the shRNA stably integrated in their genome were selected. Western blot analysis was utilised for evaluation of successful TRMT2A downregulation. Viral strains #856 and #1574 exhibited almost complete silencing of TRMT2A expression, regardless of deployed MOI. Strains #736 and
#1502 induced slight downregulation of expression, whereas transduction with strain
#1485 did not result in overt changes of expression levels. Scrambled shRNA viral transduction had no impact on TRMT2A protein levels and proved to be adequate as control. All expressional levels were compared to the amount of β–tubulin as control (Figure 20A).
Consequently, cells transduced with strains #856 and #1574 featured feasible prerequisites for further experiments regarding polyQ toxicity in a mammalian model system. Eventually cells with stable TRMT2A knockdown (derived from infection with strain #1574) were used (Figure 20B, C). Additionally, TRMT2A silencing was confirmed by mass-spectrometric analysis (see chapter 5.7).
5.6.2 Transfection of stable TRMT2A knockdown cells with polyQ constructs
For replication of Drosophila results in mammalian cells, the impact of TRMT2A knockdown on polyQ aggregation was investigated. Therefore, stably transduced HEK293 cells were transfected with different huntingtin constructs harbouring either a 25 repeats polyQ tract (GFP-HttQ25) or a pathological tract of 103 glutamines (GFP-HttQ103, both kind gift by Jan Senderek, ETH Zürich). PolyQ protein expression and aggregation could be visualised and monitored via a carboxy-terminal GFP-tag and fluorescence microscopy.
Whereas normal Huntingtin was equally distributed throughout the cytoplasm (Figure 21A, upper row), the expanded polyQ tracts rendered the protein prone to Figure 20. Stable shRNA-mediated silencing of TRMT2A expression after viral transduction of HEK293 cells.
(A) Subsequent to transduction of shRNA against TRMT2A by lentiviral particles, protein levels of viral strains #856 and #1574 were reduced most efficiently of all tested lines. Non-target shRNA control showed no marked change in TRMT2A protein levels. (B) Exemplary Western blot of decreased TRMT2A protein levels in ultimately utilised line
#1574 HEK cells compared to scrambled shRNA control. (C) Quantification of TRMT2A protein levels in line #1574 HEK cells compared to control.
t-test was used for statistics in (C), significant changes are ** p < 0.01.
A
B C
aggregation, resulting in peri- or intranuclear inclusions (Figure 21A, lower row). For expression of mutant polyQ constructs, protein aggregation or cell toxicity no obvious discrepancies could be discerned between control and knockdown cells. Nevertheless, quantification of GFP-positive cells with inclusions showed a slight, however significant increment for inclusion bodies (Figure 21B) from control cells (Figure 21A, left column) to the most potent knockdown cell line, #1574 (Figure 21A, right column).
Figure 21. Aggregation properties of normal and expanded Huntingtin in control and TRMT2A knockdown HEK cells.
(A) Transfection of HEK cells with normal GFP-tagged Huntingtin (Q25, upper row) led to cytoplasmic distribution of polyQ protein in control and knockdown cells. Expanded Huntingtin (Q103, lower row) forms prominent inclusion bodies in control and knockdown cells alike (detailed view in inset right lower row). (B) Significant increase in the fraction of transfected TRMT2A knockdown cells bearing inclusion bodies compared to control.
All scale bars in (A) apply to 50 µm. t-test was used for statistics in (B), significant changes are * p < 0.05.
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B
5.6.3 Investigation of aggregation in polyQ-transfected knockdown cells
Apart from microscopic evaluation, polyQ aggregation under the influence of TRMT2A knockdown was also investigated biochemically. Utilising the filter retardation assay, the formation of SDS-insoluble aggregates of two different huntingtin constructs was assessed in control and knockdown cells. One construct was the afore utilised exon 1 Huntingtin with a GFP-tag (GFP-HttQ103), the second one expresses Huntingtin with the 590 N-terminal amino acids and a myc-tag (myc-Htt590). As expected, non-expanded polyQ protein (in both constructs Q25) did not show increased susceptibility to aggregation and was not retained on the filter membrane neither in control nor in knockdown cells (Figure 22A). Upon GFP-HttQ103 and myc-Htt590 Q97 expression, control cells faced heavy polyQ protein aggregation. In line with the Drosophila findings, TRMT2A knockdown resulted in a significant amelioration of SDS-insoluble aggregate load in HEK293 cells trapped on the membrane (Figure 22A). Therefore, TRMT2A knockdown seems to be sufficient to significantly alleviate SDS-insoluble polyQ aggregate load in mammalian cells.
In conclusion, silencing of TRMT2A expression in mammalian cells partially was capable of recapitulating alleviating effects on SDS-insoluble polyQ aggregates. A dissolving effect like for RNAi of CG3808 affecting in situ polyQ inclusion could not be verified. Yet it remains to be solved how the consequences of TRMT2A silencing are brought about mechanistically on a molecular level.
Figure 22. Impact of TRMT2A knockdown on different SDS-insoluble Huntingtin aggregates.
(A) Q25-Huntingtin shows no SDS-insoluble protein aggregates, Q103 and Q97 proteins result in heavy aggregate load in control cells which is mitigated in TRMT2A knockdown cells. (B) Quantification of decrease in Huntingtin aggregate load in TRMT2A knockdown cells compared to control.
For protein detection in (A), mouse anti-GFP and mouse anti-myc antibodies were used. t-test was used for statistics in (B), significant changes are * p < 0.05
A B
5.7 Attempts on revelation of the molecular mechanism of TMRT2A