Investigating The Mechanisms of ELFN1 Deficiency Disorder

Introduction

ELFN1 Deficiency Disorder is a rare neurodevelopmental genetic condition. Patients with this disorder display a wide spectrum of symptoms including ADHD, ASD, epilepsy, seizures, and developmental delays.

As it is a newly identified condition there are currently gaps in our literature and understanding of it. My research aims to build foundational knowledge of the pathogenic and molecular function of this disorder.

Background

In order to function correctly, our brains require proper neuron-to-neuron communication.

The location where two neurons connect is called the synapse. ELFN1 is a specific protein that helps form the neuronal connections and binds to a glutamate receptor, called group III mGluR. Glutamate is a chemical messenger that stimulates neurons in our brain.

When ELFN1 is removed in mice there is an excess release of glutamate, which leads to hyperactivity, anxiety, and seizure susceptibility. In this situation, a drug, called positive allosteric modulator (PAM), can be implemented to bind with the group III mGluR and help control the release of glutamate (Figure 1).

As of today, 7 different genetic variants of ELFN1 have been identified in human patients. Similarly to when ELFN1 is removed in mice, these variations in the protein may disrupt neuronal communication and result in excess glutamate release in the synapse. However, in human patients it causes disorders such as seizures, epileptic encephalopathies, and ADHD. Collectively, these variants are associated with ELFN1 Deficiency Disorder.

For this project I am focusing on two variants of ELFN1, located in the N-terminal and C-terminal regions (Figure 2).

Hypothesis

I hypothesized that genetic variations in the ELFN1 protein cause the impaired ELFN1 functioning within patients harbouring the variants, leading to ELFN1 Deficiency Disorder.

Methodology

1. Western immunoblot

To assess total protein expression, HEK-293 cells were transfected with plasmids encoding ELFN1 or its variants. Next, cells were harvested, lysed, and total protein was extracted. Samples underwent SDS–PAGE followed by immunoblotting with an anti-Myc antibody. Membranes were then imaged using ImageLab 5.0 (Bio-Rad). Protein expression levels were quantified from the resulting images, and statistical analysis was performed using nonparametric one-way ANOVA. Experiments were conducted with three biological replicates (n = 3).

2. Biotinylation assay

To measure membrane protein expression, I performed a cell surface biotinylation assay. HEK293 cells were transfected with plasmids encoding ELFN1 or its variants. Cells were then harvested and incubated with biotin to label surface proteins. Following biotinylation, cells were lysed and total protein was extracted. Biotinylated proteins were isolated and underwent SDS–PAGE, followed by immunoblotting with an anti-Myc antibody. Membranes were then imaged and analyzed using ImageLab 5.0 (Bio-Rad). Protein expression levels were quantified from the resulting images, and statistical analysis was performed using nonparametric one-way ANOVA . Experiments were conducted with four biological replicates (n = 4).

3. Transcellular signalling assay

To investigate the functional effects of ELFN1 and its variants, I employed a sensor-based assay to monitor G protein activation downstream of group III mGluRs. HEK293 cells were transfected separately with plasmids encoding ELFN1 or its variants, or group III mGluRs together with the sensor. Cells were subsequently harvested and co-incubated for 1 hour to allow transcellular interactions. Afterwards, cells were stimulated with glutamate and G protein activation dynamics were measured using multimode plate reader (BMG LABTECH). Data was analyzed using nonparametric one-way ANOVA. Experiments were conducted with four biological replicates (n = 4).

Results

1. ELFN1-∆CT exhibits disrupted or low protein expression

Western blotting demonstrated that standard ELFN1 and the N-terminal variation (∆NT-ELFN1) have similar expression of proteins. However, the C-terminal variation (ELFN1-∆CT) has reduced total protein expression (Figure 4).

2. ∆NT-ELFN1 exhibits disrupted or low membrane expression

Biotinylation assay showed that standard ELFN1 and the C-terminal variation (ELFN1-∆CT) have similar membrane expressions. However, the N-terminal variation (∆NT-ELFN1) has lower membrane expression (Figure 5).

3. ELFN1-∆CT successfully downregulates receptor signalling but ∆NT-ELFN1 does not

Transcellular signaling assay revealed that as expected, standard ELFN1 cannot down-regulate group II mGluRs signalling but it can down-regulate group III mGluRs. Group II mGluRs were used as a control test.

This assay also demonstrated that the C-terminal variation (ELFN1-∆CT) can down-regulate group III mGluRs signalling, however the N-terminal variation (∆NT-ELFN1) fails to do the same (Figure 6).

For this study I analyzed my data using one-way ANOVA multiple comparison tests and t-tests in GraphPad Prism.

Discussion

The N-terminal variation (∆NT-ELFN1) has similar total expression but reduced membrane expression and cannot down-regulate group III mGluR signalling compared to standard ELFN1. This variation is unable to function properly because it has reduced membrane expression and/or might be interfering with the group III mGluR-binding in the synapse.

The C-terminal variation (ELFN1-∆CT) has reduced total expression but similar membrane expression and can down-regulate group III mGluR signalling like standard ELFN1. Although this variant is pathogenic it still functions properly, suggesting it may be disrupting intracellular signalling pathways within the synapse, resulting in epilepsy and seizures in patients.

Conclusion

This is currently ongoing research. As previous studies have found positive allosteric modulator (PAM) improve symptoms in ELFN1 knockout mice models. Extending upon this research, I propose to evaluate whether PAM or related treatments can effectively target dysfunction caused by ELFN1 variants in patients with ELFN1 Deficiency Disorder. Such strategies may offer a promising approach for symptom reduction and improved quality of life.

Next Steps

Short-term trajectory

I will conduct a co-immunoprecipitation (co-IP) assay to investigate the binding of ELFN1 variants with group III mGluRs.

I will also do immunocytochemistry (ICC) to observe the location of the protein within the cells.

Long-term trajectory

I will use differentiation of stem cells, reprogrammed from human patient blood cells, into mature neurons to observe binding patterns between ELFN1 variants and group III mGluRs through immunocytochemistry.

A mouse model is being developed to do behaviour analysis on mice with ELFN1 Deficiency Disorder and treat them with positive allosteric modulator (PAM) with the goal of improving their symptoms.

Acknowledgements

I'd first like to extend my deepest gratitude to Dr. Henry A. Dunn for providing me with the opportunity to work in his lab and expand upon his research. My sincerest thanks go towards my mentor, Afroza Parvin, as without her guidance, assistance, and supervision this work would not have been possible.

I am also appreciative of my fellow lab members for their encouragement, and to the funding agencies whose contributions made this work possible.

Lastly, I'd like to thank my parents and teachers for their unwavering support and confidence in me throughout this journey.

References

Berry-Kravis, E. M., Lindemann, L., Jønch, A. E., Apostol, G., Bear, M.F., Carpenter, R.L., Crawley, J.N., Curie, A., DesPortes, V., Hossain, F., Gasparini, F., Gomez-Mancilla, B., Hessl, D., Loth, E., Scharf, S. H., Wang, P. P., Von Raison, F., Hagerman, R., Spooren, W., & Jacquemont, S.(2018, April). Drug development for neurodevelopmental disorders: Lessons learned from Fragile X syndrome. Nature reviews. Drug Discovery. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904225/

Dore R, Chang CT, Declève A, Brunori G, Ludlam WG, Martemyanov KA, Maroofian R. ELFN1 deficiency: The mechanistic basis and phenotypic spectrum of a neurodevelopmental

disorder with epilepsy. Genet Med. 2025 Sep; 27(9): 101506. doi:10.1016/j.gim.2025.101506.

Dunn, H. A., Patil, D. N., Cao, Y., Orlandi, C., & Martemyanov, K. A. (2018). Synaptic adhesion protein ELFN1 is a selective allosteric modulator of group III metabotropic glutamate

receptors in trans. Proceedings of the National Academy of Sciences of the United States of America, 115(19), 5022–5027. https://doi.org/10.1073/pnas.1722498115

Dolan, J., & Mitchell, K. J. (2013). Mutation of ELFN1 in mice causes seizures and hyperactivity. PLOSONE, 8(11), e80491. https://doi.org/10.1371/journal.pone.0080491