Background

Disease causing variants in Proline-Rich Transmembrane Protein 2 (PRRT2) are associated with infantile seizures and/or movement disorders and are amongst the most common epilepsy risk genes (Landolfi et al. 2021, Knowles et al. 2022). PRRT2 is a synaptic protein with a putative role in negatively regulating exocytosis in neurons postnatally and is highly expressed in the human foetal brain (Mo et al. 2019, Li et al. 2016). However, neither its function in early human brain development, nor the underlying pathophysiological mechanisms associated with PRRT2 loss-of-function variants have been elucidated. Our lab has established a unique model system which allows, for the first time, a study of the developmental consequences of altered prenatal gene function in an intact, developing, human cortical network (McLeod et al. 2023). This model represents a powerful platform for investigating the effects of PRRT2.

 

Aim

Assess the impact of PRRT2 loss-of-function on early human development.

 

Methodology

Human brain slice cultures were ethically sourced from the Human Developmental Biology Resource (www.hdbr.org) in Newcastle, which provides samples of 13–18 post-conception weeks (pcw) human foetal cerebral cortex tissue. These cultures can be sustained for several weeks in vitro with gross anatomical structures maintained and new synaptic networks forming.

To assess how PRRT2 changes over time, slice cultures were fixed in 4% paraformaldehyde at different days in vitro (DIV) and labelled with antibodies against PRRT2. Different short hairpin RNA (shRNA) clones were designed to knock-down (KD) PRRT2, and their efficacy was tested in HEK293 cells using both immunocytochemistry and western blot techniques. The most efficient shRNA clone was subsequently packaged into an adeno-associated viral vector and applied to the human slice cultures. These were fixed and the impact of PRRT2 loss-of-function on the morphology and synaptic connectivity of developing neurons was evaluated.

 

Results

PRRT2 immunoreactive puncta more than doubled, between 0 and 21 DIV, in the subplate region of human cortical slice cultures (n=3 biological cultures, p<0.05, Kruskal-Wallis test, Dunn’s multiple comparison test). We established a clone that diminishes PRRT2 levels by about 50% without affecting cell integrity in HEK293 cells (n=3 passages, p<0.05, one-way ANOVA, Tukey’s post-hoc multiple comparison test), thereby mimicking the haploinsufficient state found in patients. This was further validated with application of the viral vector to the human slice cultures. The gross morphology of subplate neurons, assessed as the number and length of primary processes, was not affected by the reduction in PRRT2 levels (n=1 biological culture, 5-6 slices, unpaired t-test). However, the levels of the postsynaptic glutamatergic marker Homer1 increased following PRRT2 KD, while the presynaptic glutamatergic marker vGlut1 remained unchanged (n=1 culture, 5-6 slices, p<0.05, unpaired t-test).

 

Conclusion

This model enables us to investigate the role of PRRT2 in early brain development and assess the consequences of decreased protein levels on evolving early neuronal networks, thereby elucidating potential disease-causing mechanisms. Increased Homer1 levels after PRRT2 KD could be a result of increased neurotransmitter exocytosis. This will be further investigated using electrophysiological techniques and by increasing the number of replicates.