Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by the progressive degeneration of motor neurons in the brain and spinal cord. The cause of sporadic ALS is unknown, and there is no effective therapy. Approximately 10% of ALS cases are familial. In 1993, superoxide dismutase 1 (SOD1) was identified as the responsible gene for some cases of familial ALS (Rosen et al. 1993); since then, many other causative genes have been reported, such as TAR DNA-binding protein 43 (TDP-43), fused in sarcoma (FUS)/translocated in liposarcomas (TLS), optineurin (OPTN), C9ORF72, and profilin 1. More than 10 genes that cause familial ALS have been identified; however, among the genes for typical ALS phenotypes, OPTN is virtually the only gene in which a loss-of-function mutation is considered the principal disease mechanism.
OPTN mutations, including a homozygous deletion, a homozygous nonsense mutation, and a heterozygous missense mutation, were initially identified in Japanese ALS families (Maruyama et al. 2010). OPTN mutations have since been reported in ALS patients from many countries, especially in Europe (Belzil et al. 2011; Del Bo et al. 2011; Millecamps et al. 2011; Tumer et al. 2012).
OPTN is involved in basic cellular functions including protein trafficking, maintenance of the Golgi apparatus (Sahlender et al. 2005), and the NF-κB pathway, and ALS-related OPTN mutants were unable to suppress NF-κB activity in cultured cells (Maruyama et al. 2010). OPTN is colocalized with inclusions in motor neurons of sporadic ALS patients as well as in SOD1-, TDP-43-, or FUS-positive inclusions (Ito et al. 2011). Therefore, OPTN could function in a common pathway for ALS pathogenesis. OPTN is also colocalized with inclusion bodies in other neurodegenerative diseases such as Alzheimer's disease and Creutzfeldt-Jakob disease, suggesting its involvement in a variety of neurodegenerative processes (Osawa et al. 2011).
Recently, it was reported that the mRNA and protein levels of the p65 subunit of NF-κB are increased in the spinal cords of sporadic ALS patients and that TDP-43, which is a major pathological protein in sporadic ALS, increases the activity of NF-κB in vitro (Swarup et al. 2011).
In this study, we examined the effect of OPTN knockdown by siRNA in neuronal cells in terms of the NF-κB activity and cell viability. We also investigated the molecules downstream of NF-κB.
- Top of page
- Materials and methods
Different mutations in OPTN cause two different diseases: ALS, which results from motor neuron death, and normal pressure glaucoma, a subset of POAG (Rezaie et al. 2002), which results from the loss of retinal ganglion cells. Identification of the exact mechanisms discriminating the two allelic disorders is challenging. Based on the previous report showing that over-expression of OPTNE50K leads to retinal degeneration in mice (Chi et al. 2010), it appears that the death of RGCs by OPTNE50K, which is the most common mutation in POAG, is caused by the gain of toxic functions. However, we think that ALS is caused by the loss of OPTN function for two reasons. First, the ALS-related Q398X mutation of OPTN scarcely expresses protein because of nonsense-mediated mRNA decay. Secondly, several large deletion mutations of OPTN expected to result in null alleles were reported (Osawa et al. 2011). While there are several precedent works on OPTN knockdown in RGCs (Li et al. 2011; Sippl et al. 2011), these might not be an appropriate model for POAG, and we believe that OPTN knockdown in neuronal cells reflects pathophysiological conditions in ALS. Therefore, we used Neuro2a cells, a model cell line of motor neurons in ALS research, even though Neruo2a cells are immortal and, in some settings, the results of experiments with these cells should be carefully interpreted (LePage et al. 2005).
OPTN knockdown in mouse neuroblastoma cells increased the NF-κB activity, and cell death was induced in OPTN-knockdown cells. The rate of live cell loss was larger than the dead cell count after OPTN knockdown. There are two reasons for this apparent discrepancy. First, the trypan blue assay counts only morphologically discernible dead cells, not degraded or fragmented cell debris. Thus, the trypan blue assay might underestimate the number of dead cells. Secondly, as OPTN affects cell growth in RGC-5 cells and PC-12 cells (Li et al. 2011), the results might reflect both the cell death and the slow growth rate of Neuro2a cells.
ALS-related OPTN mutants increased NF-κB activity, but it is unknown whether increased NF-κB activity is the direct cause of the neuronal death caused by OPTN mutants. Our experiments with withaferin A clearly showed that NF-κB works in the pathway for OPTN-induced neuronal death.
NF-κB is a transcription factor that regulates hundreds of genes involved in innate immunity, cell survival and death, and inflammation, and it is involved in the production of many cytokines. We examined the NF-κB-regulated genes that were involved in cell death and survival to determine how OPTN deletion leads to neuronal death. As a result, we found that the mRNA levels of p53 and TNFα were significantly up-regulated after OPTN knockdown.
p53 is a tumor suppressor and regulates apoptosis. p53 is abnormally elevated in central nervous system lesions in sporadic ALS (Martin 2000). p53 regulates the expression of proteins found in the intrinsic mitochondrial death pathway, transcriptionally activating pro-apoptotic genes such as Bax (Miyashita and Reed 1995). We also found a tendency for Bax up-regulation after OPTN knockdown. In mutant SOD1 transgenic mice, a mouse model of ALS, Bax deletion protects motor neuron death (Gould et al. 2006). Therefore, we speculate that up-regulation of NF-κB activity after OPTN knockdown induces neuronal death via the p53-dependent mitochondrial death pathway.
TNFα is a pro-inflammatory cytokine and is increased in the blood of patients with sporadic ALS (Poloni et al. 2000). In spinal cords of mutant SOD1 transgenic mice, the mRNA and protein levels of TNFα are increased, suggesting neuroinflammation involvement for motor neuron death (Yoshihara et al. 2002; Hensley et al. 2003). The mRNA level of TNFα was up-regulated after OPTN knockdown, which may reflect neuroinflammation in vivo.
In this study, we found that neuronal cell death was induced by OPTN knockdown, which lead to NF-κB activation. Proapoptotic molecules such as p53 and Bax representing downstream targets of NF-κB are suggested to be involved in neuronal death. These results suggest that NF-κB suppression is a promising strategy for the treatment of ALS.