Hyperphosphorylation and cleavage at D421 enhance tau secretion.

It is well established that tau pathology propagates in a predictable manner in Alzheimer's disease (AD). Moreover, tau accumulates in the cerebrospinal fluid (CSF) of AD's patients. The mechanisms underlying the propagation of tau pathology and its accumulation in the CSF remain to be elucidated. Recent studies have reported that human tau was secreted by neurons and non-neuronal cells when it was overexpressed indicating that tau secretion could contribute to the spreading of tau pathology in the brain and could lead to its accumulation in the CSF. In the present study, we showed that the overexpression of human tau resulted in its secretion by Hela cells. The main form of tau secreted by these cells was cleaved at the C-terminal. Surprisingly, secreted tau was dephosphorylated at several sites in comparison to intracellular tau which presented a strong immunoreactivity to all phospho-dependent antibodies tested. Our data also revealed that phosphorylation and cleavage of tau favored its secretion by Hela cells. Indeed, the mimicking of phosphorylation at 12 sites known to be phosphorylated in AD enhanced tau secretion. A mutant form of tau truncated at D421, the preferential cleavage site of caspase-3, was also significantly more secreted than wild-type tau. Taken together, our results indicate that hyperphosphorylation and cleavage of tau by favoring its secretion could contribute to the propagation of tau pathology in the brain and its accumulation in the CSF.

The formation of tau pathological phospho-epitopes in the axon is prevented by the dephosphorylation of selective sites in primary hippocampal neurons over-expressing human tau.

In tauopathies including Alzheimer's disease, the axonal microtubule-associated protein tau becomes hyperphosphorylated at pathological epitopes and accumulates in the somato-dendritic compartment. However, it remains unclear whether tau becomes phosphorylated at these epitopes in the somato-dendritic compartment and/or in the axon. In primary hippocampal neurons where human tau was over-expressed both in the somato-dendritic compartment and the axon, the pathological epitopes recognized by the antibodies AT8 (S199/S202/T205), AT100 (T212/S214/T217), and AT180 (T231/S235) were found in the somato-dendritic compartment but not in the axon where tau was either not phosphorylated (T205 and T217) or not simultaneously phosphorylated (T231 and S235) at sites included in the above epitopes. When transfected neurons were treated with the phosphatase inhibitor, okadaic acid, AT8, AT100 and AT180 epitopes were observed in the axon, indicating that tau was dephosphorylated at selective sites of pathological epitopes in this compartment. Expression of tau mutants where one phosphorylation site included in the above epitopes was mutated in alanine showed that the formation of one of these epitopes was not required for the formation of the two others in primary hippocampal neurons. All together our results indicate that in the somato-dendritic compartment, the kinase and phosphatase activity does not prevent the formation of pathological epitopes whereas in the axon, the amount of tau phosphorylated at the pathological epitopes is regulated by phosphatase activity, most likely that of phosphoserine/phosphothreonine phosphatase 2A, the major tau phosphatase. This indicates that if the pathological epitopes are initially formed in the axon in Alzheimer's disease brain, the activation of phosphatases could be an efficient way to abolish their generation.

Application of Rho antagonist to neuronal cell bodies promotes neurite growth in compartmented cultures and regeneration of retinal ganglion cell axons in the optic nerve of adult rats.

Inactivation of Rho promotes neurite growth on inhibitory substrates and axon regeneration in vivo. Here, we compared axon growth when neuronal cell bodies or injured axons were treated with a cell-permeable Rho antagonist (C3-07) in vitro and in vivo. In neurons plated in compartmented cultures, application of C3-07 to either cell bodies or distal axons promoted axonal growth on myelin-associated glycoprotein substrates. In vivo, an injection of C3-07 into the eye promoted regeneration of retinal ganglion cell (RGC) axons in the optic nerve after microcrush lesion. Delayed application of C3-07 promoted RGC growth across the lesion scar. Application of C3-07 completely prevented RGC cell death for 1 week after axotomy. To investigate the mechanism by which Rho inactivation promotes RGC growth, we studied slow axonal transport. Reduction in slow transport of cytoskeletal proteins was observed after axotomy, but inactivation of Rho did not increase slow axonal transport rates. Together, our results indicate that application of a Rho antagonist at the cell body is neuroprotective and overcomes growth inhibition but does not fully prime RGCs for active growth.

Vaccination stimulates retinal ganglion cell regeneration in the adult optic nerve.

We examined whether vaccination of adult rats with spinal cord homogenate (SCH) can promote regeneration of retinal ganglion cells (RGCs) after microcrush lesion of the optic nerve. Injured animals vaccinated with SCH showed axon growth into the optic nerve and such regeneration was not observed in animals vaccinated with liver homogenate (LH). Regeneration was not a consequence of neuroprotection since our vaccine did not protect RGCs from axotomy-induced cell death. Sera of vaccinated animals were tested for antibodies against myelin-associated glycoprotein, NogoA, Nogo-66 receptor, or chondroitin sulphate proteoglycans (CSPG), but no significant levels were detected. Antibodies to myelin basic protein were present in the serum of some SCH-vaccinated animals. In culture, serum from SCH-vaccinated animals promoted RGC growth on myelin but not on CSPG. Our results show that the effect of the pro-regenerative vaccine is mediated by antibodies to SCH. However, we were not able to detect a significant immune reaction to growth inhibitory proteins, suggesting alternative mechanisms for the success of vaccination to promote regeneration.