Role of the sortilin gene family in major depressive disorder

Abstract

Major Depressive Disorder (MDD) is a leading cause of disability and a major contributor to the global burden of disease. Nearly 300 million people are affected by depression, a statistic projected to rise due to the effects of the COVID19 pandemic. Although pharmacological intervention exists in the form of antidepressants, antidepressant therapy will only work for approximately one third of patients. A further third will experience relief of symptoms only after trial and error of multiple antidepressants and the remaining third of patients will suffer from treatment-resistant depression. The limited efficacy of antidepressants is thought to be due to their actions being indirect. The majority act by increasing monoamine levels, an effect which although immediate, is followed by delayed therapeutic onset. Despite the global burden of MDD, the pathogenesis is not fully understood. The discovery of more efficacious antidepressants is dependent upon identifying molecular targets.

The sortilin family is comprised of five type-I transmembrane receptors; sortilin (encoded by the SORT1 gene), SorLA (encoded by the SORL1 gene), SORCS1, SORCS2 and SORCS3. All share the characteristic N-terminal vacuolar protein sorting 10 protein (Vps10p) binding domain which allows for the binding and trafficking of a variety of ligands such as neuropeptides and neurotrophins. In this way, sortilins play a role in neuronal viability and function. They regulate protein transport and signal transduction and are vital for growth and maturation of neurons and synapses, and for synaptic plasticity in the developing and adult brain.

Genome-wide association studies (GWAS), as well as human and rodent functional studies have implicated two members of the sortilin family (SORCS3 and sortilin) in MDD. SORCS3 is the top/one of the top hits in multiple GWAS strongly suggesting a role in the pathogenesis of MDD. The functions of SORCS3 have mainly been investigated through mouse models. The dynamic expression of Sorcs3 in the developing brain compared to its restricted pattern in the adult brain suggests it may be required for morphogenic signalling events such as neurogenesis. SORCS3 is also important for synaptic plasticity. Sorcs3 deficient mice exhibit a loss of long-term depression and defects in fear memory.

In order to investigate the functions of SORCS3, I used genome editing to knock-out the gene in two human induced pluripotent stem cell lines. I then differentiated the lines into neural precursor cells and forebrain neurons in order to model some of the key events that occur during human embryonic and adult neurogenesis in vitro. I found that a lack of SORCS3 may lead to differences in proliferation and viability of NPCs as well as neural differentiation (e.g. reduced detection of immature neuronal markers such as doublecortin and βIII-tubulin and increase in the mature marker NeuN) and potential differences in the rates of spontaneous apoptosis. Interestingly, this was only the case for one of the two cell lines used in this project. This could be due to experimental or donor variation, which is commonly reported in the literature for iPSC lines, and highlights the importance of reproducing results in independent lines. This, and other studies, demonstrated differences in the expression level of SORCS3 between the two cell lines, even at the iPSC stage. A difference in the expression of the protein of interest could explain differences in the impact of knocking the gene out. A limitation of this study was the small number of clones used for each line and so larger numbers will be needed to confirm these potential deficits.

Both mouse and human functional studies also implicate sortilin, encoded by the SORT1 gene, in MDD. There is contradictory evidence regarding whether increased or decreased levels of sortilin are associated with MDD. For example, sortilin was upregulated in the neocortex and hippocampus of chronically stressed mice and was associated with depression-like behaviour. Sortilin expression was also reduced in depression-resistant rats. Studies in patients with MDD have also shown increased expression of sortilin which was downregulated following treatment with antidepressants. In contrast, decreases in the sortilin propeptide have been detected in patients with MDD, which increase in response to treatment with antidepressants or ECT. Decreases in sortilin also lead to increased anxiety-like behaviour in mice.

Linked to the pathogenesis of MDD are increased oxidative stress, DNA damage and impaired antioxidant defence. A recent role has been shown for the sortilin family member Sorcs2 in the protection of neurons from oxidative stress. As members of the sortilin family can have overlapping functions, I investigated the role of sortilin in oxidative stress, DNA damage and antioxidant defence in neurons. Using CRISPR-Cas9 genome editing, I knocked out SORT1 in the human dopaminergic neuronal cell line LUHMES. I induced oxidative stress using hydrogen peroxide and found reduced viability in SORT1 KO neurons. DNA double strand breaks were visualised using immunostaining for the marker ɣH2AX. I did not find any difference in the number of DSBs between WT and KO at baseline or after induced oxidative stress however the number of lines used in these experiments was low. I then measured the expression of antioxidant enzymes after induction of oxidative stress and found lower expression in SORT1 KO neurons. This result, combined with the finding that SORT1 expression was also differentially expressed in response to oxidative stress in WT lines, could point to a role for sortilin in oxidative stress that future experiments could further elucidate.

In conclusion, this project comprises the first efforts towards examining the function of SORCS3 in human cell lines. It has also suggested a role for sortilin in antioxidant defence. Difficulties with maintaining LUHMES neurons in culture for a sufficient length of time may have impeded this work which may have been helped by switching to another cell line. Although I generated SORT1 KO iPSC lines, due to COVID restrictions and time restraints I was unable to investigate this avenue. Further research with these lines could lead to greater insight into the cellular functions of sortilin and SORCS3 within the context of MDD which could translate to new therapeutic targets in the future.