My research focuses on neurodegeneration, with a particular focus on Amyotrophic Lateral Sclerosis (ALS). ALS is a progressive and fatal disorder involving the degeneration of motor neurons, eventually resulting in muscle paralysis and death, often within five years of diagnosis. Despite decades of research, we still do not fully understand the molecular mechanisms driving neuronal death, and the few approved treatments prolong survival by mere months. Although approximately 10% of ALS cases are inherited via rare mutations, the vast majority of ALS is considered sporadic (sALS). The causes of sALS are complex and poorly understood, although a combination of genetic and environmental factors likely play a role.

Interestingly, a common cellular feature seen in > 97% of ALS cases is the mislocalisation of the nuclear protein TDP-43 into the neuronal cytoplasm. Cytoplasmic TDP-43 readily becomes phosphorylated and forms insoluble aggregates, interfering with important cellular processes. Crucially, mislocalised TDP-43 can no longer fulfil its nuclear function of regulating the splicing of important neuronal genes. This may result in the inclusion of “cryptic exons” in the mature mRNA, often causing loss of function via nonsense-mediated decay.

Unfortunately, there is a lack of robust cellular (in vitro) models available to study the molecular consequences of TDP-43 mislocalisation. Current models are limited due to their reliance on rare mutated forms of the TDP-43 gene, its overexpression to abnormal levels, or the use of toxic chemicals to induce mislocalisation. We therefore began by developing an improved cellular model of TDP-43 proteinopathy.

METHODOLOGY:

We used CRISPR-Cas9 editing to tag endogenous TDP-43 with GFP in healthy human induced pluripotent stem cells (iPSCs). Furthermore, we employ a GFP-specific nanobody with a strong nuclear export signal (NES) which will strongly bind TDP43-GFP and export it from the nucleus into the cytoplasm.

We differentiate these iPSCs into motor neurons and then induce TDP-43 mislocalisation via transduction with adeno-associated viruses (AAVs) that express the nanobody-NES sequence. Our controls express the nanobody without any NES. This method therefore does not require any exogenous TDP-43 expression, mutation, or chemical addition to induce mislocalisation.

RESULTS:

A. We can trigger TDP-43 mislocalisation in our model on demand. TDP-43 localisation is unchanged in the controls (no NES) but becomes cytoplasmic upon expression of the NES nanobody. Scale bar = 50 µm.

B. Mislocalisation of TDP-43 in our model captures splicing defects reported in the literature. Loss of nuclear TDP-43 causes abnormal inclusion of cryptic exons in important neuronal genes UNC13A and STMN2, with subsequent reduction of their canonical transcripts. CE = cryptic exon, TRUNC = truncated STMN2 (caused by CE inclusion). Results show qPCR data from 3 independent differentiations, and error bars show SEM. ** = p < 0.01, *** = p < 0.001.

FUTURE WORK:

I hope to use this model to study the impact of TDP-43 mislocalisation on RNA and RNA-binding protein (RBP) homeostasis. We are now delving into single-cell RNA sequencing to uncover RNA splicing changes in various neuronal subtypes. It would be great to know if some neuronal subtypes are more susceptible to certain cryptic splice variants than others and whether this contributes to motor neuron-specific vulnerability in ALS. Furthermore, I would like to investigate the consequence of cryptic splicing of key RBPs on motor neuron survival and morphology.

FUNDED BY:

University of Exeter, Medical Research Council

CONTACT:

jg576@exeter.ac.uk

Johanna Ganssauge

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