Death Receptor6and Caspase6Regulate PrP106-126-induced Axonal Degeneration In Rat Spinal Neurons

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are fatal neurodegenerative disorders, characterized by brain vacuolation, neuronal cell death and microgliosis. They are caused by the conversion of cellular prion protein (PrPC) into the pathological isoform (PrPSc) through conformational changes. PrPSc is protease-resistant, and has a higher proportion of β-sheet structure in place of the normal a-helix structure. Axonal degeneration is a hallmark of many neurodegenerative disorders. It has also been reported that amyloid fibrils of mammalian prion protein induced axonal degeneration in NTERA2-derived terminally differentiated neurons.However, the full complement of axonal degeneration triggers is not fully understood. DR6is a death domain-containing TNF receptor (TNFR). Like other TNFR family members, DR6is widely expressed by neurons as neurons differentiate and enter a pro-apoptotic state. We hypothesized that axonal degeneration occurs in prion diseases and DR6participates in the degenerative process of axons. To test our hypothesis, we investigated axonal alterations of neuron and the roles of DR6and apoptotic factors, caspase3and caspase6, in axonal degeneration in rat spinal neurons incubated with PrP106-126. The results showed that:1. The cytoskeleton of spinal cord neurons was severely damaged after treatment with PrP106-126. There was loss of axons in most of the spinal neurons, and the microtubule (tubulin) of the distal end of remaining axons appeared filamentous fractured and protein of tubulin aggregation was also evident. We believe that PrP106-126triggered axonal degeneration of spinal cord neurons by damaging the microtubule.2. While massive neuronal deaths and axonal degeneration occurred in PrP106-126-treated, DR6expression increased significantly in PrP106-126-treated neurons. The knockdown of DR6protected neurons from PrP106-126-induced degeneration. Our findings indicate that axonal degeneration of neurons is regulated by DR6.3. Fluorescence examination showed that both caspase3and caspase6were activated in PrP106-126-treated neurons. Interestingly, caspase3was highly enriched in cell bodies, with no expression in axons and caspase6was expressed in both cell bodies and axons. The caspase3inhibitor blocked cell body but not axons degeneration in PrP106-126-treated cultures and the caspase6inhibitor blocked the degeneration of both cell body and axons. Our findings suggest that caspase6regulated axonal degeneration induced by PrP106-126.4. Caspase6activation was inhibited by DR6siRNA; no caspase6was activated in neurons transfected with DR6siRNA and then treated with PrP106-126. Similarly, caspase3activation was also inhibited by DR6siRNA. The results support an activation model of apoptotic signaling pathway that caspase6is downstream of DR6and both DR6and caspase6are modulators of axon degeneration.5. Caspase6activation in Prp106-126-treated cells was unaffected by preincubation with Nmnatl. Nmnat did not protect axonal degeneration induced by PrP106-126after incubation for24hours. However, it protected neurons from axonal degeneration within12hours. The results indicated that although Nmnat did not block caspase6activation after PrP106-126treatment, but it delayed axonal degeneration.