Amyotrophic lateral sclerosis (ALS) affects millions of people worldwide, and till now there is no cure for this disease. ALS is characterized by degeneration of motor neurons either in the brain or in the spinal cord or both and is followed by the death of motor neurons (Brown and Al-Chalabi, 2017). Based on genetic heritability, ALS is classified as familial ALS (fALS) if family history is established, and sporadic ALS (sALS) otherwise. A clear cause for the onset of sALS is unknown; however, many studies have revealed that the mutation in certain specific genes is associated with sALS. It has been documented that 50 genes are involved in causing ALS (Mejzini et al., 2019). In this paper, we examine the role of one of these 50 genes, optineurin (OPTN), in maintaining the balance between the apoptosis, necroptosis and autophagy in association with other regulatory factors.

OPTN gene was discovered as one of the marker's for ALS in 2010, and is located in chromosome 10 (Maruyama et al., 2010). It is expressed in the brain, heart, kidney, liver, placenta, pancreas and human skeletal muscle (Li et al., 1998). The presence of OPTN in almost all organs underscores its importance in humans. OPTN is a highly conserved hexameric 64 kDa protein, consisting of 577 amino acids. The post-translational phosphorylation and ubiquitination of this protein play an important role in its structural and functional stability (Ying et al., 2010). It acts as a multifunctional protein and mediates different cellular processes through its binding with different partners. All OPTN-binding proteins bind to specific domains and perform downstream functions such as autophagy, apoptosis, necroptosis, inflammatory response, vesicular trafficking, and vasoconstriction. For instance, the Tank Binding Kinase-1(TBK1) binds and phosphorylates OPTN (Richter et al., 2016), and thereby helps in the regulation of autophagy.

BDNF which is known for its regulation of apoptosis, plays a key role in survival and growth of neurons. Jiang et al. (2016) have shown that RE1 silencing transcription factor (REST) and CoREST negatively regulate BDNF while miRNA-9 (miR-9) is responsible for post-transcriptional repression of REST gene (Wu and Xie, 2006) and CoREST gene (Packer et al., 2008). In a study related to necroptosis, it was reported that OPTN activates receptor-interacting protein kinase 1 (RIPK1) which then activates receptor-interacting protein kinase 3 (RIPK3) through phosphorylation. RIPK3 in turn activates mixed-lineage kinase-domain like pseudo-kinase (MLKL) through phosphorylation (Ito et al., 2016). It has been reported that MLKL activates the chemical factors which regulate the cell death pathway. Also, there is evidence that decrease in the level of OPTN also decreases the level of RIPK1, RIPK3, and MLKL mRNA and protein (Ito et al., 2016). In concert with apoptosis and necroptosis, autophagy helps in the maintenance of neuronal cells by regulation of organellar and biomolecular homeostasis through degradation and recycling processes. Here also, OPTN plays an important role and disturbances in autophagy has been linked to ALS (Liu et al., 2014; Ying and Yue, 2016). Tank binding kinase (TBK1) binds to OPTN and mediates its phosphorylation. Subsequently, SQSTM1 (P62) which is also a marker of autophagy, binds to OPTN which in turn promotes binding of light-chain 3B (LC3) to the LC3-interacting (LIR) domain of OPTN and regulates autophagy (Madill et al., 2017).

Twenty five OPTN variants, mostly of the missense type, are involved in ALS and glaucoma disease (Toth and Atkin, 2018). OPTN-E478G and OPTN-E50K, their effect on the regulation of cell-death processes have been reported in the literature. Over expression of OPTN-E478G causes increasing of pro inflammatory cytokines in OPTN-E478G expressing mouse embryonic fibroblast cell and it leads to neuronal cell death (Liu et al., 2018). In OPTN-E50K mutation, the brain-derived neurotrophic factor (BDNF) level decreases in retinal ganglionic cell and causes neurodegeneration (Jiang et al., 2016). Our group discovered a novel mutation OPTN-K489E in OPTN gene while conducting targeted next-generation sequencing of DNA samples isolated from the blood samples of Indian ALS patients (Narain et al., 2018). This mutation was found in 2/154 Indian ALS patients having the site of onset in bulbar and upper limb region. ALS-functional-rating-score (ALSFRS) of the two patients were 28 and 21. As the pathogenicity of this mutation had not been reported, the details of its mechanisms of action and elucidation of participating pathways will require a host of experimental evidence. In this work, we have investigated the effect of OPTNK489E mutation on apoptosis, necroptosis, and autophagy, and how it causes death in SH-SY5Y neuronal cell. Differentiated SH-SY5Y cells shows phenotypic neurons, however, it is reported that differentiation of SH-SY5Y can change several miRNA expressions which controls many arresting factors like REST and CoREST (Das and Bhattacharyya, 2014). Since the aim of this study included miRNA9, REST and CoREST, we used undifferentiated SH-SY5Y cells for our investigation. We have studied the effect of OPTN K489E on the gene and protein expression of REST, Co-REST, BDNF, RIPK1, RIPK3, MLKL, P62, LC3II, and TBK1, and determined a plausible mechanism of the neuronal cell death in ALS patient having OPTN mutation. Overall, we have carried out a comparative study of the effect of OPTN-WT and OPTN-K489E mutation in SH-SY5Y cells and present our results on miR9 and BDNF-mediated apoptosis, RIPK1, RIPK3, and MLKL-mediated necroptosis, and P62–LC3II mediated autophagy. This work elucidates the possible role of OPTN and OPTN-K489E in neuronal cell death regulation and confirms pathogenicity of the OPTN-K489E mutation.