The Pathogenesis Of FUS Protein Disease

Since the discovery that mutations in FUS (fused in sarcoma) gene are associated with familial or sporadic ALS and FTLD, this gene has drawn considerable attention of researchers on its role in neurodegeneration. FUS proteinopathy is defined as a spectrum of diseases possessing similar pathological features, namely the hallmark intracellular inclusion bodies immunoreactive for FUS antibody, including BIBD, FTLD-FUS and ALS6. Intensive investigations on prevalence and pathological features of FUS proteinopathy have been made. However, a detailed diagram of cellular events and specific pathways involved in disease progression remains largely unclear.To begin to understand the underlying mechanisms of FUS proteinopathy, we made transgenic flies expressing wild-type FUS or ALS-linked mutants, R524S and P525L. When expressed in a variety of fly neurons, wild-type and mutant FUS caused progressive degeneration, with the mutants exhibiting more potent neurotoxicity. These phenotypes include progressive eye degeneration, loss of mushroom body neuron axons, morphological abnormalities of motoneurons and locomotion defects.The mitochondria in FUSWt and FUSP525L expressing fly larval motor axons were found smaller then control group, implicating altered balance of mitochondrial fission-fusion as a result of FUS expression. We then carried out systematic characterizations, and confirmed occurrence of mitochondrial dysfunction, which was revealed by loss of membrane potential and over-production of ROS.Following morphological examinations, we conducted live imaging to monitor dynamic mitochondrial trafficking within the axons of fly motoneurons expressing FUSWt or FUSP525L, and found that bi-directional mitochondrial trafficking was disturbed remarkably. In detail, axonal mitochondria spent less time moving but more time pausing; during the motile phase mitochondria ran slower than control with reduced processivity. In contrast, the frequencies of pauses and reversals were significantly higher. With regard to the above parameters, mutant led to more severe deficits than wild-type FUS. Notably, the retrograde transport was preferentially affected by FUS expression.We hypothesized that such defects were due to mitochondrial dysfunction, since it is believed that axonal transport slow down when mitochondrial depolarization occurs. We demonstrated that when FUSWt or FUSP525L was overexpressed, PINK1 accumulated and recruited Parkin, which is an E3 ligase targeting multiple mitochondrial proteins, implicating activation of mitochondrial quality control. Outer-membrane protein MIRO1, a receptor for motor binding and a target of Parkin, was found to be hyper-ubiquitinated, while MIR02 ubiquitination level stayed the same.According to the above results, we speculated that it was the MIRO1 hyper-ubiquitination that halted mitochondria due to loss of available receptor. We verified the involvement of these genes in pathogenesis using fly genetic screening with eye degeneration as the readout. Knock-down of Pink1 or Park partially rescued eye degeneration, and greater improvement was achieved when both of them were down-regulated, which proved that they play pathogenic roles in disease. However, overexpression or knock-down Miro had no effect on eye phenotype. Manipulation of Miro level in fly motoneurons gave different implications, that co-expression of Miro and mutant FUS led to declined larval viability and motility; whereas knock-down of Miro partially rescued mutant induced locomotion impairment. Prompt by such discovery, we began to check the axonal trafficking in the above flies. Down-regulation of Miro reverted FUS impaired axonal transport to normal level in both directions, as could be seen in prolonged motile phase and shortened stationary phase, as well as velocity similar to control mitochondria. Restoration of mitochondrial transport was also demonstrated by longer runs, shorter pauses, and lower frequencies of pauses and reversals in Miro silenced larvae.In conclusion, we have established a powerful animal model for FUS proteinopathy. It is demonstrated that FUS proteinopathy is accompanied by disturbance in axonal transport of mitochondria; however, restoration of transport could not block but delay neurodegeneration. The relationship of defective axonal transport to ALS is still under debate. This finding provides evidence for a pathogenic role of transport disturbance in pathogenesis of neurodegeneration caused by FUS mutation, suggesting that it participate and accelerate the disease progression.