PFTK1 kinase regulates axogenesis during development via RhoA activation.

Cyclin Dependent Kinase family members include members of the non-cell cycle CDK, such as PFTK1/Eip63E. Eip63E expresses primarily in postnatal and adult nervous system in Drosophila melanogaster but its role in CNS development remains unknown. We sought to understand its role in the CNS by studying the fly ventral nerve cord during development. Eip63E regulates axogenesis in neurons and its deficiency leads to neuronal defects. We describe a functional interaction between Eip63E and Rho1. Studies in cultured cortical neurons from PFTK1 knockout mice, confirmed that PFTK1 plays a role in axonal outgrowth and its deficiency resulted in faster growing axons. We demonstrate that GDP bound RhoA is a substrate of PFTK1 and this phosphorylation resulted in higher activity of RhoA. In conclusion, our work represents the first steps in the characterization of the neuronal functions of PFTK1 and points to RhoA activation in the regulation of PFTK1 mediated axogenesis.

Transthyretin Promotes Axon Growth via Regulation of Microtubule Dynamics and Tubulin Acetylation

Transthyretin (TTR), a plasma and cerebrospinal fluid protein, increases axon growth and organelle transport in sensory neurons. While neurons extend their axons, the microtubule (MT) cytoskeleton is crucial for the segregation of functional compartments and axonal outgrowth. Herein, we investigated whether TTR promotes axon elongation by modulating MT dynamics. We found that TTR KO mice have an intrinsic increase in dynamic MTs and reduced levels of acetylated α-tubulin in peripheral axons. In addition, they failed to modulate MT dynamics in response to sciatic nerve injury, leading to decreased regenerative capacity. Importantly, restoring acetylated α-tubulin levels of TTR KO dorsal root ganglia (DRG) neurons using an HDAC6 inhibitor is sufficient to completely revert defective MT dynamics and neurite outgrowth. In summary, our results reveal a new role for TTR in the modulation of MT dynamics by regulating α-tubulin acetylation via modulation of the acetylase ATAT1, and suggest that this activity underlies TTR neuritogenic function.

Cytoskeletal assembly in axonal outgrowth and regeneration analyzed on the nanoscale

The axonal cytoskeleton is organized in a highly periodic structure, the membrane-associated periodic skeleton (MPS), which is essential to maintain the structure and function of the axon. Here, we use stimulated emission depletion microscopy (STED) of primary rat cortical neurons in microfluidic chambers to analyze the temporal and spatial sequence of MPS formation at the distal end of growing axons and during regeneration after axotomy. We demonstrate that the MPS does not extend continuously into the growing axon but develops from patches of periodic β-spectrin II arrangements that grow and coalesce into a continuous scaffold. We estimate that the underlying sequence of nucleation, elongation, and subsequent coalescence of periodic β-spectrin II patches takes around 15 hours. Strikingly, we find that development of the MPS occurs faster in regenerating axons after axotomy and note marked differences in the morphology of the growth cone and adjacent axonal regions between regenerating and unlesioned axons. Moreover, we find that inhibition of the spectrin-cleaving enzyme calpain accelerates MPS formation in regenerating axons and increases the number of regenerating axons after axotomy. Taken together, we provide here a detailed nanoscale analysis of MPS development in growing axons.

Injury-Induced HSP27 Expression in Peripheral Nervous Tissue Is Not Associated with Any Alteration in Axonal Outgrowth after Immediate or Delayed Nerve Repair

We investigated injury-induced heat shock protein 27 (HSP27) expression and its association to axonal outgrowth after injury and different nerve repair models in healthy Wistar and diabetic Goto-Kakizaki rats. By immunohistochemistry, expression of HSP27 in sciatic nerves and DRG and axonal outgrowth (neurofilaments) in sciatic nerves were analyzed after no, immediate, and delayed (7-day delay) nerve repairs (7- or 14-day follow-up). An increased HSP27 expression in nerves and in DRG at the uninjured side was associated with diabetes. HSP27 expression in nerves and in DRG increased substantially after the nerve injuries, being higher at the site where axons and Schwann cells interacted. Regression analysis indicated a positive influence of immediate nerve repair compared to an unrepaired injury, but a shortly delayed nerve repair had no impact on axonal outgrowth. Diabetes was associated with a decreased axonal outgrowth. The increased expression of HSP27 in sciatic nerve and DRG did not influence axonal outgrowth. Injured sciatic nerves should appropriately be repaired in healthy and diabetic rats, but a short delay does not influence axonal outgrowth. HSP27 expression in sciatic nerve or DRG, despite an increase after nerve injury with or without a repair, is not associated with any alteration in axonal outgrowth.

Gold and Cobalt Oxide Nanoparticles Modified Poly-Propylene Poly-Ethylene Glycol Membranes in Poly (ε-Caprolactone) Conduits Enhance Nerve Regeneration in the Sciatic Nerve of Healthy Rats

Reconstruction of nerve defects is a clinical challenge. Autologous nerve grafts as the gold standard treatment may result in an incomplete restoration of extremity function. Biosynthetic nerve conduits are studied widely, but still have limitations. Here, we reconstructed a 10 mm sciatic nerve defect in healthy rats and analyzed nerve regeneration in poly (e-caprolactone) (PCL) conduits longitudinally divided by gold (Au) and gold-cobalt oxide (AuCoO) nanoparticles embedded in poly-propylene poly-ethylene glycol (PPEG) membranes (AuPPEG or AuCoOPPEG) and compared it with unmodified PPEG-membrane and hollow PCL conduits. After 21 days, we detected significantly better axonal outgrowth, together with higher numbers of activated Schwann cells (ATF3-labelled) and higher HSP27 expression, in reconstructed sciatic nerve and in corresponding dorsal root ganglia (DRG) in the AuPPEG and AuCoOPPEG groups; whereas the number of apoptotic Schwann cells (cleaved caspase 3-labelled) was significantly lower. Furthermore, numbers of activated and apoptotic Schwann cells in the regenerative matrix correlated with axonal outgrowth, whereas HSP27 expression in the regenerative matrix and in DRGs did not show any correlation with axonal outgrowth. We conclude that gold and cobalt-oxide nanoparticle modified membranes in conduits improve axonal outgrowth and increase the regenerative performance of conduits after nerve reconstruction.

Preclinical assessment on neuronal regeneration in the injury-related microenvironment of graphene-based scaffolds

As the application of graphene nanomaterials gets increasingly attractive in the field of tissue engineering and regenerative medicine, the long-term evaluation is necessary and urgent as to their biocompatibility and regenerative capacity in different tissue injuries, such as nerve, bone, and heart. However, it still remains controversial about the potential biological effects of graphene on neuronal activity, especially after severe nerve injuries. In this study, we establish a lengthy peripheral nerve defect rat model and investigate the potential toxicity of layered graphene-loaded polycaprolactone scaffold after implantation during 18 months in vivo. In addition, we further identify possible biologically regenerative effects of this scaffold on myelination, axonal outgrowth, and locomotor function recovery. It is confirmed that graphene-based nanomaterials exert negligible toxicity and repair large nerve defects by dual regulation of Schwann cells and astroglia in the central and peripheral nervous systems. The findings enlighten the future of graphene nanomaterial as a key type of biomaterials for clinical translation in neuronal regeneration.

Water Treadmill Training Ameliorates Neurite Outgrowth Inhibition Associated With NGR/RhoA/ROCK by Inhibiting Astrocyte Activation Following Spinal Cord Injury

Background: Axons become scattered and incomplete after spinal cord injury (SCI). Cross-talk between astrocytes and neurons plays a pivotal role in neurite outgrowth following SCI. Rehabilitative training is a recognized method for the treatment of SCI, but the specific mechanism of its effect on axonal outgrowth in the central nervous system (CNS) has not been determined.Methods: A total of 160 adult male SD rats weighing 200–250 g were randomly divided into three groups, and an SCI animal model was established. Rats were subjected to water treadmill training (TT) for 7 or 14 d. The Basso-Beattie-Bresnahan (BBB) motor function scale, hematoxylin-eosin (HE) staining, Nissl staining, Western blotting and immunofluorescence were used to measure the degree of neurological deficit, tissue morphology, quantitative expression and accurate localization of the corresponding proteins.Results: We found that TT decreased tissue structure damage and improved functional recovery. TT promoted the regeneration of neurons and reduced apoptosis induced by SCI around the lesion. TT significantly increased the expression of GAP43 and NF200 after SCI. In addition, the injury-induced increase in the expression of proinflammatory factors was significantly inhibited by TT. TT reduced the activation of astrocytes and microglia, accompanied by reduced expression of C3d and higher increased of S100A10. Finally, the level of chondroitin sulfate proteoglycan (CSPG) surrounding the lesion and activation of the NGR/RhoA/ROCK signalling pathway in neurons after SCI were effectively inhibited by TT.Conclusions: In this study, we found that TT played a novel role in recovery from SCI by promoting axonal outgrowth associated with the NGR/RhoA/ROCK by inhibiting astrocyte activation after SCI.

Glycosylation in Axonal Guidance

How millions of axons navigate accurately toward synaptic targets during development is a long-standing question. Over decades, multiple studies have enriched our understanding of axonal pathfinding with discoveries of guidance molecules and morphogens, their receptors, and downstream signalling mechanisms. Interestingly, classification of attractive and repulsive cues can be fluid, as single guidance cues can act as both. Similarly, guidance cues can be secreted, chemotactic cues or anchored, adhesive cues. How a limited set of guidance cues generate the diversity of axonal guidance responses is not completely understood. Differential expression and surface localization of receptors, as well as crosstalk and spatiotemporal patterning of guidance cues, are extensively studied mechanisms that diversify axon guidance pathways. Posttranslational modification is a common, yet understudied mechanism of diversifying protein functions. Many proteins in axonal guidance pathways are glycoproteins and how glycosylation modulates their function to regulate axonal motility and guidance is an emerging field. In this review, we discuss major classes of glycosylation and their functions in axonal pathfinding. The glycosylation of guidance cues and guidance receptors and their functional implications in axonal outgrowth and pathfinding are discussed. New insights into current challenges and future perspectives of glycosylation pathways in neuronal development are discussed.