CNS axons globally increase axonal transport after peripheral conditioning.

Despite the inability of CNS axons to regenerate, an increased regenerative capacity can be elicited following conditioning lesion to the peripheral branch of dorsal root ganglia neurons (DRGs). By in vivo radiolabeling of rat DRGs, coupled to mass spectrometry and kinesin immunoprecipitation of spinal cord extracts, we determined that the anterograde transport of cytoskeleton components, metabolic enzymes and axonal regeneration enhancers, was increased in the central branch of DRGs following a peripheral conditioning lesion. Axonal transport of mitochondria was also increased in the central branch of Thy1-MitoCFP mice following a peripheral injury. This effect was generalized and included augmented transport of lysosomes and synaptophysin- and APP-carrying vesicles. Changes in axonal transport were only elicited by a peripheral lesion and not by spinal cord injury. In mice, elevated levels of motors and of polyglutamylated and tyrosinated tubulin were present following a peripheral lesion and can explain the increase in axonal transport induced by conditioning. In summary, our work shows that a peripheral injury induces a global increase in axonal transport that is not restricted to the peripheral branch, and that, by extending to the central branch, allows a rapid and sustained support of regenerating central axons.

Neuronal deletion of GSK3ß increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2.

BACKGROUND: In the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity. RESULTS: Following a phospho-site broad signaling pathway screen, we found that in conditioned neurons with high regenerative capacity, decreased glycogen synthase kinase 3ß (GSK3ß) activity and increased microtubule growth speed in the growth cone were present. To investigate the importance of GSK3ß regulation during axonal regeneration in vivo, we used three genetic mouse models with high, intermediate or no GSK3ß activity in neurons. Following spinal cord injury, reduced GSK3ß levels or complete neuronal deletion of GSK3ß led to increased growth cone microtubule growth speed and promoted axon regeneration. While several microtubule-interacting proteins are GSK3ß substrates, phospho-mimetic collapsin response mediator protein 2 (T/D-CRMP-2) was sufficient to decrease microtubule growth speed and neurite outgrowth of conditioned neurons and of GSK3ß-depleted neurons, prevailing over the effect of decreased levels of phosphorylated microtubule-associated protein 1B (MAP1B) and through a mechanism unrelated to decreased levels of phosphorylated cytoplasmic linker associated protein 2 (CLASP2). In addition, phospho-resistant T/A-CRMP-2 counteracted the inhibitory myelin effect on neurite growth, further supporting the GSK3ß-CRMP-2 relevance during axon regeneration. CONCLUSIONS: Our work shows that increased microtubule growth speed in the growth cone is present in conditions of increased axonal growth, and is achieved following inactivation of the GSK3ß-CRMP-2 pathway, enhancing axon regeneration through the glial scar. In this context, our results support that a precise control of microtubule dynamics, specifically in the growth cone, is required to optimize axon regrowth.

HDAC-6 inhibition ameliorates the early neuropathology in a mouse model of Krabbe disease.

Introduction: In Krabbe disease (KD), mutations in ß-galactosylceramidase (GALC), a lysosomal enzyme responsible for the catabolism of galactolipids, leads to the accumulation of its substrates galactocerebroside and psychosine. This neurologic condition is characterized by a severe and progressive demyelination together with neuron-autonomous defects and degeneration. Twitcher mice mimic the infantile form of KD, which is the most common form of the human disease. The Twitcher CNS and PNS present demyelination, axonal loss and neuronal defects including decreased levels of acetylated tubulin, decreased microtubule stability and impaired axonal transport. Methods: We tested whether inhibiting the α-tubulin deacetylase HDAC6 with a specific inhibitor, ACY-738, was able to counteract the early neuropathology and neuronal defects of Twitcher mice. Results: Our data show that delivery of ACY-738 corrects the low levels of acetylated tubulin in the Twitcher nervous system. Furthermore, it reverts the loss myelinated axons in the sciatic nerve and in the optic nerve when administered from birth to postnatal day 9, suggesting that the drug holds neuroprotective properties. The extended delivery of ACY-738 to Twitcher mice delayed axonal degeneration in the CNS and ameliorated the general presentation of the disease. ACY-738 was effective in rescuing neuronal defects of Twitcher neurons, stabilizing microtubule dynamics and increasing the axonal transport of mitochondria. Discussion: Overall, our results support that ACY-738 has a neuroprotective effect in KD and should be considered as an add-on therapy combined with strategies targeting metabolic correction.

Myelin Lipids Inhibit Axon Regeneration Following Spinal Cord Injury: a Novel Perspective for Therapy.

Lack of axon regeneration following spinal cord injury has been mainly ascribed to the inhibitory environment of the injury site, i.e., to chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors (MAIs). Here, we used shiverer (shi) mice to assess axon regeneration following spinal cord injury in the presence of MAIs and CSPG but in the absence of compact myelin. Although in vitro shi neurons displayed a similar intrinsic neurite outgrowth to wild-type neurons, in vivo, shi fibers had increased regenerative capacity, suggesting that the wild-type spinal cord contains additional inhibitors besides MAIs and CSPG. Our data show that besides myelin protein, myelin lipids are highly inhibitory for neurite outgrowth and suggest that this inhibitory effect is released in the shi spinal cord given its decreased lipid content. Specifically, we identified cholesterol and sphingomyelin as novel myelin-associated inhibitors that operate through a Rho-dependent mechanism and have inhibitory activity in multiple neuron types. We further demonstrated the inhibitory action of myelin lipids in vivo, by showing that delivery of 2-hydroxypropyl-ß-cyclodextrin, a drug that reduces the levels of lipids specifically in the injury site, leads to increased axon regeneration of wild-type (WT) dorsal column axons following spinal cord injury. In summary, our work shows that myelin lipids are important modulators of axon regeneration that should be considered together with protein MAIs as critical targets in strategies aiming at improving axonal growth following injury.