Loss of UNC13A: how it exacerbates amyotrophic lateral sclerosis, and how to correct it

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a devastating neurodegenerative disorder that causes progressive loss of MNs, leading to impaired muscle function and paralysis. ALS is incurable and leads to death, usually caused by the inability to breathe, on average only 3 years after diagnosis, with a lifetime risk of about 1 in 400.

A crucial player in the disease mechanism of ALS is a protein called TDP-43. TDP-43 mislocalisation in neurons occurs in ~97% of ALS, regardless of its many possible causes. TDP-43 is important for RNA splicing, a process by which RNA, the information deriving from DNA, is cut and re-assembled to provide cells with essential information. When TDP-43 cannot perform its function, numerous alterations of splicing occur, including a phenomenon called "cryptic exons", where novel random sequences are inserted into RNAs.

In ALS brains, following TDP-43 alterations, multiple cryptic exon mistakes occur, and it has been difficult to understand which ones play a role in disease. We have now identified a novel cryptic exon event in the UNC13A gene. UNC13A encodes a protein that is important for the functioning of synapses, which allow neurons to transfer information between each other - the essential step for brain function.

Unexpectedly, what makes the UNC13A cryptic exon different from the numerous other cryptic exons, is that genetics data from ALS patients supports it being very important for disease. In fact, a common genetic change in UNC13A has been long known to be associated with ALS, but why has remained elusive.

Here, with the discovery of the UNC13A cryptic exon, we have been able to provide the missing link. In summary, when TDP-43 alterations occur in ALS, they induce the UNC13A cryptic exon, which in turn leads to a reduction of this crucial synaptic protein.

Our hypothesis is that preventing this loss will have a positive impact on ALS disease course.

In this research proposal we aim to better understand the UNC13A cryptic exon in ALS, its consequences and how we can prevent it from happening. We will:

1. Identify in exactly what neuronal subtypes UNC13A is altered in ALS. This question is important to both understand the disease, and to know what we need to target in therapeutic interventions.
We will answer our question by studying brains and spinal cords from ALS cases. We will visualise the altered UNC13A RNA and also use novel techniques that allow us to study all the RNAs in multiple single neurons from these cases.

2. Investigate how the UNC13A cryptic exon impacts basic neuronal functions. This work will shed new light on disease mechanism in ALS and will be crucial to set up future assays for therapeutic development.
We routinely use human (iPSC-derived) ALS cortical and motor neurons in culture and we can induce the UNC13A cryptic exon. We will study, with microscopy and electrophysiology, how the synapse functions in these neurons, and we will also study if other neuronal problems associated with ALS are impacted by the UNC13A cryptic exon.

3. Understand the mechanism underlying the UNC13A cryptic exon and devise strategies to prevent it. This will allow us to understand how the UNC13A cryptic exon occurs, and will allow us to design oligonucleotide strategies - currently being used in the clinic for other neurological diseases - to interfere with this toxic mechanism.
We have developed small versions of UNC13A that allow us to study the splicing mechanism very effectively. We will use this system also to efficiently screen and identify the best oligonucleotides for preventing this event.

In summary, this project will contribute to understanding how changes in UNC13A, induced by TDP-43, impact on neurons and ALS pathogenesis, regardless of underlying cause. Crucially, this project will also develop an approach to prevent these changes and perform the initial steps for a potential therapeutic strategy for ALS.

The RNA-binding protein TDP-43 is a central player in ALS and other neurodegenerative disorders. It accumulates in cytoplasmic inclusions and is depleted from the nucleus, where it normally resides to regulate fundamental processes including RNA splicing. As a consequence, widespread splicing changes occur in ALS, including the appearance of cryptic exons (CE): novel toxic intronic sequences incorrectly incorporated into mature RNA.

Many CEs have been described and their role in pathology, if any, is largely obscure. However, we have identified a novel CE in the synaptic gene UNC13A that is incorporated due to TDP-43 depletion in neurons. Crucially, a genetic risk variant for ALS lies within this CE. We have shown this SNP enhances the CE inclusion. The CE causes a reduction of UNC13A RNA and protein; this event is specific for brain regions most affected in ALS.

Thus, we hypothesise that UNC13A loss contributes to ALS pathogenesis and rescuing this may lead to amelioration of disease, potentially in ~97% of ALS with TDP-43 mislocalisation.

We propose to investigate fundamental questions about UNC13A misprocessing in ALS, and how to correct it.
We will further characterise WHERE the alterations occur in ALS brains, using single molecule RNA visualisation techniques and single neuronal nuclear sequencing to identify affected neuronal populations. We will use our established iPSC-derived cortical and lower motor neurons, in which we reliably recapitulate TDP-43 mediated loss of UNC13A, to understand WHAT the consequences are for neuronal biology-by analysing ALS phenotypes such as neuronal survival, neurite and axon growth, and with specific synaptic electrophysiology investigations. Lastly, we will determine HOW the UNC13A CE is regulated, and develop approaches to interfere with it, and rescue downstream consequences.

These investigations will shed light on a crucial ALS pathogenesis step and set the basis for a novel therapeutic approach for ALS.