We recently revealed that the axon endoplasmic reticulum resident transcription factor Luman/CREB3 (herein called Luman) serves as a unique retrograde injury signal in regulation of the intrinsic elongating form of sensory axon regeneration. Here, evidence supports that Luman contributes to axonal regeneration through regulation of the unfolded protein response (UPR) and cholesterol biosynthesis in adult rat sensory neurons. One day sciatic nerve crush injury triggered a robust increase in UPR-associated mRNA and protein expression in both neuronal cell bodies and the injured axons. Knockdown of Luman expression in 1 d injury-conditioned neurons by siRNA attenuated axonal outgrowth to 48% of control injured neurons and was concomitant with reduced UPR- and cholesterol biosynthesis-associated gene expression. UPR PCR-array analysis coupled with qRT-PCR identified and confirmed that four transcripts involved in cholesterol regulation were downregulated >2-fold by the Luman siRNA treatment of the injury-conditioned neurons. Further, the Luman siRNA-attenuated outgrowth could be significantly rescued by either cholesterol supplementation or 2 ng/ml of the UPR inducer tunicamycin, an amount determined to elevate the depressed UPR gene expression to a level equivalent of that observed with crush injury. Using these approaches, outgrowth increased significantly to 74% or 69% that of injury-conditioned controls, respectively. The identification of Luman as a regulator of the injury-induced UPR and cholesterol at levels that benefit the intrinsic ability of axotomized adult rat sensory neurons to undergo axonal regeneration reveals new therapeutic targets to bolster nerve repair.SIGNIFICANCE STATEMENTThe endoplasmic reticulum (ER) plays a vital role in the sensing and responding to cellular perturbations through alterations in protein and cholesterol biosynthesis, with quality control being ensured through the unfolded protein response (UPR). But the UPR's role in sensory axon regeneration is largely unknown. In sensory axons, the ER generates an important retrograde injury signal through the synthesis and cleavage/activation of the ER transmembrane transcription factor, Luman/cAMP response element binding protein 3, that is retrogradely transported to the nucleus where it regulates the intrinsic of ability of these neurons to regenerate an axon. We now show that Luman does so by regulating the UPR and cholesterol biosynthesis at levels that are beneficial to axon regeneration.

Significance: Peripheral nerve injury often occurs at axonal sites remote from the neuronal cell bodies. Information about these remote injuries must be accurately relayed back to the cell body as part of the reprogramming that occurs to transition the neuron to a regenerating state. We provide new insights into how the axonal endoplasmic reticulum (ER) proximal to the injury site generates a critical regeneration signal in sensory neurons. This involves injury-triggered activation and synthesis of an intraaxonal, ER transmembrane transcription factor, Luman/cAMP response element binding protein 3, that is retrogradely transported back into the nucleus. Manipulation of axon-derived Luman expression in injured sensory neurons reveals an important role for Luman in regulation of the intrinsic elongating form of axonal growth associated with the regeneration state.

Rapid and efficient axon remyelination aids in restoring strong electrochemical communication with end organs and in preventing axonal degeneration often observed in demyelinating neuropathies. The signals from axons that can trigger more effective remyelination in vivo are still being elucidated. Here we report the remarkable effect of delayed brief electrical nerve stimulation (ES; 1 hour @ 20 Hz 5 days post-demyelination) on ensuing reparative events in a focally demyelinated adult rat peripheral nerve. ES impacted many parameters underlying successful remyelination. It effected increased neurofilament expression and phosphorylation, both implicated in axon protection. ES increased expression of myelin basic protein (MBP) and promoted node of Ranvier re-organization, both of which coincided with the early reappearance of remyelinated axons, effects not observed at the same time points in non-stimulated demyelinated nerves. The improved ES-associated remyelination was accompanied by enhanced clearance of ED-1 positive macrophages and attenuation of glial fibrillary acidic protein expression in accompanying Schwann cells, suggesting a more rapid clearance of myelin debris and return of Schwann cells to a nonreactive myelinating state. These benefits of ES correlated with increased levels of brain derived neurotrophic factor (BDNF) in the acute demyelination zone, a key molecule in the initiation of the myelination program. In conclusion, the tremendous impact of delayed brief nerve stimulation on enhancement of the innate capacity of a focally demyelinated nerve to successfully remyelinate identifies manipulation of this axis as a novel therapeutic target for demyelinating pathologies.