Investigating the relationship between SORCS2 and SORLA and DNA double-stranded break formation, as a possible mechanism in neuropsychiatric disorders
Item statusRestricted Access
Embargo end date27/06/2021
Gospodinova, Katerina Ognyanova
SORCS2 and SORLA are members of the Vps10p-domain receptor family, which comprises five multifunctional neuronal receptors. All family members are involved in intracellular sorting and trafficking of various neurotrophic factors and their precursor forms, as well transmembrane receptors and synaptic proteins. This gene family has been implicated in a broad range of cellular processes, including neuronal health, differentiation and synaptic plasticity. Importantly for this thesis, SORLA and SORCS2 have been shown to play a role (SORLA in vivo and in vitro; SORCS2 in vitro) in the processing of the amyloid precursor protein (APP) and thus amyloid β (Aβ) production. Genetic and functional studies provide further evidence for the involvement of both receptors, alongside other family members, in cognition and various brain disorders. DNA damage and compromised DNA repair have been implicated in brain aging, psychiatric and neurodegenerative disorders. Recently, a specific type of DNA damage- DNA double- stranded break (DSB) formation, has been implicated in both Alzheimer’s disease (AD) and physiological brain activity, i.e. learning and memory processes. In mice, exploration of a novel environment led to a transient increase in DSB formation in the dentate gyrus (an area of the brain important for learning and memory). The breaks were repaired within 24 hours. Subsequently, it was shown that these breaks are generated by Topoisomerase II β (TopoIIβ) and are required for the expression of early-response genes. In mice, pathological Aβ levels were associated with increased neuronal DSB formation and repair deficits. The effect of Aβ on DSB formation and repair was attributed to aberrant network activity and dysregulated NMDA receptor signalling. Given the link between sortilins and neuropsychiatric illness, as well as their role in APP processing and, recently, NMDA receptor trafficking (SorCS2), my project aimed to address the relationship between the two genes and DSB formation. I examined levels of DNA DSBs in WT and Sorcs2-/- mice before and after exploration of a novel environment. Following exploratory activity, WT mice showed increased numbers of DSBs, which were repaired within 24 hours. Meanwhile, I detected elevated levels of DSBs at baseline in the dentate gyrus of Sorcs2-/- mice. Moreover, compared to the corresponding WT mice, the Sorcs2-/- mice exhibited an altered response to the novel environment, as they failed to acquire new breaks. To explore possible mechanisms that could explain this phenotype, I knocked out SORCS2 in the human dopaminergic neuronal cell line LUHMES using CRISPR/Cas9 genome editing. I did not observe any difference in the extent of DSB formation in untreated SORCS2 knockout (KO) clones, compared to WT controls grown simultaneously. However, SORCS2 KO neurons showed elevated levels of DSBs following treatment with etoposide- a compound that prevents the re-ligation of TopoIIβ-induced DSBs. In addition, knocking out SORCS2 was also associated with reduced neuronal viability. I next explored possible mechanisms underlying the observed increase in etoposide induced DSBs in the SORCS2 KO clones. In line with previous work, treatment with NMDA led to a significant increase in the number of DSBs in WT LUHMES neurons. I did not, however, observe any difference in the extent of DSB formation in NMDAtreated SORCS2 KO clones compared to WT controls. This experiment was performed on a small number of WT and KO clones and the results may thus reflect lack of power to detect a difference. However, if this lack of difference was confirmed in subsequent experiments, this would suggest that the increase in etoposide-induced DSBs in the SORCS2 KO clones is unlikely to be due to dysregulated NMDA signalling. Meanwhile, I was unable to measure Aβ42 levels reliably in LUHMES. Thus, the possible role of Aβ in the increased DSB formation observed in etoposide-treated SORCS2 KO clones cannot be excluded. Surprisingly, mouse primary hippocampal neurons derived from Sorcs2-/- pups did not show any difference in DSB levels with or without treatment with etoposide. However, this experiment was performed on a limited number of hippocampal cultures and thus the results obtained may not reflect a true lack of difference. The possible discrepancy between the results in the etoposide-treated primary hippocampal and LUHMES dopaminergic neurons, but more importantly the role of SorCS2 in dopaminergic signalling, led me to investigate whether knocking out SORCS2 alters extracellular dopamine levels, as a potential mechanism underlying the increased DSB formation. ELISA analysis showed no difference in dopamine release between SORCS2 KO and WT LUHMES neurons. Due to technical difficulties and time constraints, I was unable to assess the levels of DSB formation in Sorl1-/- mice or primary hippocampal neurons derived from them. Obtaining SORL1 KO LUHMES cell lines using CRISPR/Cas9 genome editing also proved to be challenging. However, I generated mutant cell lines carrying the rare SORL1 missense mutation, G508S, which is associated with early onset AD. Despite being located at a splice site, introducing the G508S mutation in LUHMES did not affect splicing. However, it led to reduced SORL1 mRNA, but not protein, levels. In conclusion, the work completed in this project constitutes the first evidence for a potential role of the sortilins in DSB formation. Future work investigating this link further might provide us with valuable knowledge on the cellular mechanisms underlying neurodegenerative and psychiatric conditions.