The Role of Stress Granules in TDP-43 Transgenic Mouse Models of FTD/ALS

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterised by the degeneration and death of upper and motor lower neurons, leading to paralysis and eventual death (Hardiman et al., 2017). The pathological hallmark of ALS is the formation of motor neuron/glial insoluble inclusions, which in ~97% patients predominantly consist of the RNA-binding protein TDP-43 (Neumann et al., 2006). TDP-43 is recruited to Stress Granules (SGs), transient membrane-less organelles that form in the presence of environmental stressors that have been linked to ALS/FTD. SGs promote cell survival through recruitment and protection of untranslated mRNAs, alongside various RNA-binding proteins (RBPs) (Taylor et al., 2016). Debate exists as to whether SGs seed TDP- 43 aggregation or are neuroprotective.

Disease causative TDP-43 mutants have been identified which enhance protein cleavage, aggregation, and neurotoxicity (Sreedharan et al., 2008). TDP-43 mutations have been utilised to generate transgenic in vivo (rodent) models of disease that recapitulate multiple features of ALS. These include mice expressing ALS-linked Q331K mutant TDP-43 (TDP-43Q331K) or co-expressing this mutant alongside human wild-type TDP-43 (TDP-43WTxQ331K) (Mitchell et al., 2015).

The organotypic slice culture (OSC) model is an ex vivo model where both brain and spinal cord slices can be cultured (Croft et al., 2019). This model has previously been reported, but not as a model of ALS-linked pathology (Moudio et al., 2022). In this thesis I have utilised mouse cortical and lumbar OSCs to examine ALS-linked mutant TDP-43 pathology and its interaction with heat-shock induced SGs.

I report that OSCs from TDP-43WT (expressing wild-type human TDP-43), TDP-43Q331K and TDP-43WTxQ331K mice replicate pathology seen in vivo (Mitchell et al., 2015), specifically showing cytoplasmic p62 and ubiquitin positive TDP-43 inclusions. These increased in number in OSCs from TDP-43Q331K mice, and to a greater extent, TDP-43WTxQ331K (relative to NTg OSCs). I also report an OSC model for response to acute heat stress, with G3BP1-positive, TIA-1 positive and eIF4G-postitive SG formation peaking at 8 hours and dissolving after 48 hours recovery, relative to <15 minutes (min) for formation, and ~80 min for dissolution reported previously in cellular models (Wheeler et al.,
2016).

Using proximity-ligation assay (PLA), I demonstrated that key SG nucleators are separately involved in the formation of SGs, with TIA-1 acting as an early initial nucleator, then G3BP and eIF4G being recruited later. In the presence of mutant TDP-43Q331K, OSCs showed persistent SGs despite no increase in SG/TDP-43 colocalisation. There were no other changes in SG dynamics or marker interactions relative to OSCs from NTg OSCs. Finally, I showed the first successful adeno-associated virus (AAV) transduction of cortical OSCs.

These results show that the OSC model is a useful model for studying ALS-linked pathology. Furthermore, my results also suggest that the SG response to heat shock in OSCs is characterised by a longer timeline as compared to monolayer cell cultures traditionally used for stress response studies. The persistence of SGs in the presence of Tg TDP-43Q331K, alongside a lack of increased colocalisation of mutant TDP-43 with SGs, suggests induction of a chronic stress state by mutant TDP-43 (Ratti et al., 2020) and a possible protective role of SGs harbouring solely endogenous TDP-43. The transduction of OSCs with an AAV could, in future, help explore G3BP knockdown in OSCs expressing TDP-43Q331K (and other mutants), to determine impact on TDP-43 aggregation.

Despite the above conclusions, it should be noted that there are significant limitations to some of the data presented in this Thesis, in particular the technical drawbacks of the quantification approach used throughout the study. Therefore, the findings presented in this Thesis should be considered preliminary as they require further validation using complementary approaches in the future.


Date of Award1 May 2023
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorChristopher Shaw (Supervisor) & Jackie Mitchell (Supervisor)

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