The ALS-linked protein optineurin normally shields oligodendrocytes from a form of programmed cell death, and thus keeps axons tightly wrapped in insulating myelin and healthy, according to a study in the August 5 issue of Science. Optineurin also stifles inflammation in microglia, the authors report. The findings point to a potential pathogenic mechanism by which optineurin mutations could cause ALS, by allowing axon degeneration and inflammation. Furthermore, given that optineurin is found enmeshed in protein aggregates in several other neurodegenerative diseases, the authors suggest that loss of optineurin function could contribute to axonal pathology more broadly than just in ALS.
Specifically, optineurin suppresses a kinase signaling cascade that promotes cell death by necroptosis. “Inhibition of the kinase may be therapeutic in ALS,” according to principal investigator Junying Yuan of Harvard Medical School in Boston.
How optineurin loss-of-function mutations causes ALS has not been clear, Yuan said. Previous work identified the protein as a regulator of autophagy, but in an apparently redundant role (see Oct 2014 news), and thus it has been hard to establish how its absence leads to neuronal cell death.
Yuan became interested in optineurin as a result of her discovery of necroptosis, a caspase-mediated death pathway that is an alternative to apoptosis, as well as her discovery of receptor-interacting kinase 1 (RIPK1) as a key mediator of this pathways (Degterev et al., 2005; Degterev et al., 2008). But because neuronal damage typically results from both apoptosis as well as necroptosis, she said, “We struggled for a long time about the pathophysiologic significance of this pathway.” They found a clue to the importance of necrotopsis and a key mediator of the pathway, in 2008, when Yuan’s siRNA screen for sensitizers to necroptosis turned up the ALS gene optineurin (Hitomi et al., 2008).
To investigate optineurin’s role in necroptosis, Yuan, first author Yasushi Ito, and colleagues developed an optineurin knockout mouse. They found that loss of optineurin did not affect the number of motor neurons, but that beginning at 3 weeks, there was a marked demyelination of motor axons. By 12 weeks, the authors began to detect a statistically significant reduction in the number of motor neuron axons. In addition, in the ventrolateral spinal cord axons that remained alive and insulated, they saw abnormal myelination, such as a decompaction of the myelin sheath, due to death and regeneration of oligodendrocytes “The phenotype is actually subtle,” Yuan said. “I think many people have missed this because oligodendrocytes can regenerate, so you still see myelination, but it is immature.”
In order to identify where optineurin expression was most crucial, Ito knocked it out in different cell types. Selective optineurin knockouts in oligodendrocytes alone, or in myeloid cells, reproduced the loss of axons and abnormal myelination, while knockouts in astrocytes or motor neurons did not, suggesting that the critical effect is in oligodendrocytes and myeloid cells, Yuan said.
In keeping with an increase in necroptosis, expression of RIPK1 and two other mediators of necroptosis, RIPK3 and mixed lineage kinase domain-like (MLKL), were elevated in optineurin null mouse spinal cord. The levels of ubiquitinated RIPK1 were also reduced, indicating it was not being degraded. Further evidence for a link between optineurin and RIPK1 turnover was revealed by attenuated degradation of RIPK1 in mouse embryonic fibroblasts from optinuerin null mice, as compared to cells from wild-type mice. In microglia, which do not express high levels of MLKL, RIPK1 activation promoted inflammatory signaling by increasing expression of pro-inflammatory cytokines and TNF-alpha. These effects could be blocked by treatment with necrostatin-1 (Nec-1s), a specific inhibitor of RIPK1 (Degterev et al., 2008).
Yuan concluded that optineurin normally promotes RIPK1 degradation, thus suppressing necroptosis. The absence of optineurin, as occurs in some cases of ALS, allows RIPK1 levels to rise, which causes inflammation by microglia, the necroptosis of oligodendrocytes, and dying back of unmyelinated axons.
Next, the authors wanted to investigate how optineurin and RIPK1 contribute to ALS, by examining their role in ALS model mice. In the spinal cord of SOD1-G93A mice, RIPK1 and other necroptosis mediators were elevated, and administration of Nec-1s delayed axon demyelination. In addition, administration of Nec-1s beginning at 8 weeks of age delayed onset of motor dysfunction in the SOD1-G93A mice. Preliminary examination of a pathological sample from a human sporadic ALS spinal cord showed demyelination of lower spinal cord white matter, compared to a control cord sample. Immunoblotting of samples from 13 patients revealed elevated levels of RIPK1, RIPK3 and MLKL in human ALS spinal cord as compared to 10 controls.
The significance of these findings may extend well beyond ALS due to optineurin mutation, Yuan speculated. Optineurin is an ubiquitin binding protein, and is thus recruited to aggregating proteins in all forms of ALS, which might keep it from its normal protective role, and allow RIPK1 activation.
“This paper provides new insights to how optineurin prevents axon degeneration,” according to Yimin Zou of the University of California at San Diego, who was not involved in the study. “The fact that necrostatin-1 can protect axons and delay onset of motor symptoms in SOD1-G93A mice is exciting as this may lead to novel therapy.” The Nec-1s compound developed by Yuan has been licensed by her institute to a biotechnology company for clinical development, Yuan said.
Since optineurin is also found in aggregates in Alzheimer’s disease, Parkinson’s disease, and several other proteinopathies, a RIPK inhibitor may have potential as a therapy beyond ALS as well, according to Yuan. “Aggregate-trapped optineurin as a means towards functional optineurin deficiency and induction of necroptotic signaling is an attractive hypothesis that is worthy of further study in ALS and other neurodegenerative diseases,” agreed Mark Albers of Massachusetts General Hospital in Boston, MA, who was not involved in the study. The challenge, he said, will be designing the clinical trials of the RIPK1 inhibitor to intervene early enough in the disease course to have a clinically meaningful outcome.
Ito Y, Ofengeim D, Najafov A, Das S, Saberi S, Li Y, Hitomi J, Zhu H, Chen H, Mayo L, Geng J, Amin P, DeWitt JP, Mookhtiar AK, Florez M, Ouchida AT, Fan JB, Pasparakis M, Kelliher MA, Ravits J, Yuan J. RIPK1 mediates axonal degeneration by promoting inflammation and necroptosis in ALS. Science. 2016 Aug 5;353(6299):603-8.[Pubmed].
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