A glaucoma‐ and ALS‐associated mutant of OPTN induces neuronal cell death dependent on Tbk1 activity, autophagy and ER stress

Abstract

Mutations in OPTN are associated with glaucoma, an eye disease, and also with amyotrophic lateral sclerosis (ALS), a motor neuron disease. A 2‐bp insertion in OPTN (691_692insAG or 2bpIns‐OPTN) is associated with both glaucoma and ALS. This mutation results in frame shift after 127 amino acids, giving rise to a protein with C‐terminal aberrant sequence. We have explored the mechanism of induction of cell death by this mutant in a motor neuron cell line, NSC‐34, and also in a retinal cell line, 661W. Compared to wild‐type OPTN, this mutant induced more cell death in NSC‐34 and 661W cells. This mutant localizes predominantly in the nucleus whereas normal OPTN localizes in the cytoplasm. Deletion analysis of 2bpIns‐OPTN showed that the aberrant sequence was not essential for cell death induction. This mutant interacts with TANK‐binding kinase 1 (Tbk1) but not with OPTN and activates Tbk1. This mutant induced ER stress in NSC‐34 cells as seen by induction of C/EBP homologous protein (CHOP) and some other genes. Induction of CHOP, autophagosomal protein LC3‐II and cell death by this mutant were abrogated by Tbk1 knockdown and also by 4‐phenylbutyric acid, that inhibits ER stress. Induction of CHOP and cell death by 2bpIns‐OPTN was autophagy dependent as shown by the effect of Atg5 knockdown. This mutant caused increased formation of LC3‐positive aggregates. Treatment of cells with autophagy inducer rapamycin reduced LC3‐positive aggregates, CHOP and cell death induced by 2bpIns‐OPTN. These results suggest that constitutive activation of Tbk1 by 2bpIns‐OPTN leads to impaired autophagy that results in ER stress and cell death.

Crosstalk between endoplasmic reticulum stress response and autophagy in human diseases

“…Although it is not a common mutation, a two-base pair insertion in the exon 6 of the OPTN gene is associated with ALS. In NSC-34 cells harboring this mutation (a cellular model of ALS), the sustained activity of TANK-binding kinase 1 is induced, and the formation of LC3-positive aggregates is increased (Medchalmi et al 2021 ). Excessive formation of LC3-positive aggregates mediates ER stress and eventually induces apoptosis.…”

“…Excessive formation of LC3-positive aggregates mediates ER stress and eventually induces apoptosis. Another ALS-associated mutation, the TAR DNA binding protein 43 mutation (TDP-43A315 T), was found to increase ER stress markers including CHOP, GRP78, and eIF2α, as well as autophagy markers such as Beclin-1 and LC3-PE (Medchalmi et al 2021 ). Excessive neuronal damage occurs when cells with this mutation are treated with 3-MA, indicating that autophagy acts as a protective mechanism to reduce neuronal toxicity (Wang et al 2015 ).…”

Potential roles of the endoplasmic reticulum stress pathway in amyotrophic lateral sclerosis

“…Moreover, treatment with an ER stress inhibitor decreased ER stress-related gene expression and reduced cell death. 2 bpIsn-OPTN is predominantly localized in the nucleus, in contrast to wild-type OPTN mainly localized in the cytoplasm ( Medchalmi et al, 2021 ). Loss of OPTN in mouse and MEF cells also induced upregulation of ER stress pathway ( Ramachandran et al, 2021 ).…”

Autophagy in glaucoma pathogenesis: Therapeutic potential and future perspectives

“…M98K is a gain-of-function mutation, overexpression of M98K shows enhanced autophagic signaling leading to endocytic recycling of transferrin receptor (TFRC) degradation and causing cell death ( Sirohi et al, 2013 ). Uncontrolled activation of TBK1 by the mutation that A 2-bp insertion in OPTN leads to impaired autophagy that results in the accumulation of LC3-positive aggregates, which induces ER stress and a retinal cell line, 661 W, cell death ( Medchalmi et al, 2021 ). In addition, duplication of TBK1 can cause glaucoma, for the reason that increased transcription of TBK1 encodes a kinase that phosphorylates OPTN, which recruits LC3B and initiates autophagy ( Tucker et al, 2014 ).…”