Understanding the impacts of CACNA1A/unc-2 mutations on presynaptic function in C. elegans

Editor’s note: The research described below comes from a recipient of a 2024 MSC Travel Grant supporting travel to the 2024 C. elegans Topic Meeting: Neuronal Development, Synaptic Function and Behavior. These grants reimburse travel expenses for those who have had their abstract for a presentation or poster accepted at a meeting.

By  Michael Krawchuk, MD-PhD candidate, Albert Einstein College of Medicine, US.

What is the research gap that your study addresses?

Mutations in CACNA1A, which encodes the Cav2.1α1 subunit of P/Q type voltage-gated calcium channels, cause debilitating channelopathies. Cav2.1 cerebellar somato-dendritic pacemaking dysfunction leads to ataxia. However, the channel is also expressed presynaptically throughout the CNS and the main mediator of neurotransmitter release, yet the impact CACNA1A mutations have on synaptic transmission is largely unexplored. Prior work in heterologous systems lack in vivo context, while mammalian in vivo approaches are too complex to study impacts on specific cell compartments. My project aims to investigate the gap in knowledge regarding how CACNA1A mutations impact synaptic transmission using a simple in vivo approach.

What is your research hypothesis?

Patient presentation of CACNA1A mutations is complex, ranging from motor symptoms such as ataxia, to migraine and intellectual disability. I hypothesize that CACNA1A mutations leading to migraine/intellectual disability, as opposed to ataxia/motor symptoms linked to cerebellar cell body dysfunction, severely impact presynaptic structure and function, causing alterations in synaptic transmission.

What methodology did you use to address your research hypothesis?

My project uses C. elegans, a powerful in vivo genetic model with a single Cav2α1 family homolog, UNC-2, which has significant similarity to Cav2.1 and is localized exclusively to presynaptic compartments. C. elegans’ fast life cycle, genetic accessibility, and single synapse acuity allows for the generation of a variety of CACNA1A mutations and assessment of their impact on synaptic transmission in a straightforward model system. To interrogate synaptic function, we use the acetylcholinesterase inhibitor aldicarb, which leads to paralysis in worms from over-activation of acetylcholine receptors, with a time course that is dependent on the amount of synaptic vesicle release. Changes in vesicle release will then be verified with in vivo electrophysiological recordings. In addition, we use endogenous markers of the UNC-2 channel to assess expression and clustering at the presynaptic terminal. Furthermore, worm locomotion is assessed using computer software that tracks movement, and any isolated phenotypes can be subjected to a forward genetic modifier screen, probing molecular pathways further. Lastly, neural circuits impacted by CACNA1A/unc-2 mutations will be assessed via calcium imaging for changes in synaptic transmission.

What are the main results of your study?

Using aldicarb, I have shown migraine- and intellectual disability (ID)-associated mutations elicit gain-of-function (GOF) vesicle release behavior while ataxia-associated mutations don’t have an effect. In vivo electrophysiological recordings have confirmed increased excitatory, but not inhibitory, vesicle release frequency for the GOF mutants. Localization of the subunit to the presynaptic membrane appears decreased in all mutants when imaged. Lastly, only ID-associated mutations exhibit a hyper-reversal phenotype. Analyzing the effects of the ID mutations on neural circuits and downstream pathways are being explored with calcium imaging and a forward genetic screen, where two suppressors have been identified so far.

What conclusions did you reach based on your results?

In conclusion, our results indicate CACNA1A/unc-2 mutations alter synaptic transmission, channel expression, neural circuit function and behavioral output differentially based on patient presentation.

What are the limitations of your study?

The main limitation is using C. elegans to model patient mutations with complex behaviors such as migraine and intellectual disability in this simple genetic system. The behavioral repertoire of roundworms is minimal, so we cannot examine complex effects on behavior, and there are only 3 calcium channel subtypes compared to 10 in humans. Although, we are not trying to prove the mutations cause those phenotypes, rather we are focusing directly on their synaptic function effects. Additionally, the synaptic vesicle release machinery proteins and overall process is extremely homologous to mammals, bolstering the use of this model to assess synaptic transmission.

What is the relevance of your study to migraine?

One of the canonical CACNA1A disorders is familial hemiplegic migraine type 1 (FHM1). Patients suffer from partial body paralysis and frequent migraines. Furthermore, over 50% of patients with a CACNA1A mutation who have another diagnosis, such as episodic ataxia, still experience migraines. Therefore, migraines are a predominant patient symptom stemming from mutations in CACNA1A, where understanding the in vivo synaptic deficits related to migraine-associated patient mutations will help us better understand the molecular dysfunction related to migraine and work towards therapeutic targets for suffering patients. We are currently modeling two migraine-associated patient mutations, with plans to make more.