Crosstalk of TDP-43 and Optineurin in in vitro and ex vivo Models of Amyotrophic Lateral Sclerosis

Abstract

Abstract Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease marked by protein aggregation and neuroinflammation. Protein aggregates in >95% of ALS cases are cytoplasmic and contain ubiquitinated and phosphorylated TAR DNA binding protein 43 kDa (TDP-43). Mutations in a ubiquitin-binding adaptor protein optineurin have recently been found in a subset of ALS cases. Its mutations are thought to act by loss of function, leading to disbalanced inflammatory signaling and/or impaired disposal of aggregated proteins by autophagy. Here we addressed the putative link between TDP-43 proteinopathy and loss of optineurin and/or its function. To this end we generated optineurin knockout (KO) neuronal and microglial cell lines by CRISPR/Cas9 technology, and (2) primary cells from a mouse optineurin insufficiency model lacking the ubiquitin-binding region (Optn470T). Elevated TDP-43 protein levels were found in optineurin-deficient BV2 microglial cell line, and in primary Optn470T bone marrow-derived macrophages (BMDM) and microglia. No differences were detected at TDP-43 mRNA levels, arguing for post-translational regulation. Elevated TDP-43 protein levels were not caused by autophagy blockade, and were specific to myeloid cells as they were not observed in neuronal NSC-34 and Neuro2A optineurin-deficient cell lines. Upon lipopolysaccharide (LPS) stimulation, TDP-43 levels increased in WT BV2 cells and BMDM, but in BV2 KO and OPTN470T BMDM they remained at the same elevated state as in basal conditions. Our results show that optineurin directly influences basal TDP-43 protein levels in myeloid but not in neuronal cells. Further studies are needed to determine if this could be a mechanistic link to protein aggregation in ALS.