Neutrophil peptide 1 accelerates the clearance of degenerative axons during Wallerian degeneration by activating macrophages after peripheral nerve crush injury

Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.

What we know about axons in Parkinson’s disease

Tremendous research efforts have been made regarding the pathogenesis of Parkinson’s disease(PD).However,there are still no effective strategies to restore midbrain dopaminergic(mDA)innervation and prevent disease progression.One possibility is that we may have been neglecting the role of axons in mDA neuronal degeneration.This review first summarizes mDA axon development during the early stage of PD and discusses how axon guidance defects contribute to PD vulnerability.Furthermore,we review axonal transport dysregulation in the numerous PD-related genetic mutations,including Parkin,PINK1,DJ1,LRRK2 and SNCA.The evidence suggests that proper axonal transport is crucial for neuronal function and survival.Finally,advanced tools for axonal studies were evaluated,including light-sheet and super-resolution microscopy.These adapted microscopes have been used to help solve questions unanswered before.Overall,the role of axon terminals in the initiation of the degeneration cascade remains undeciphered,and more research in the related area may be conducted further to restore dopamine levels in the striatum to alleviate the motor complications of PD.

Changes in Synapses and Axons Demonstrated by Synaptophysin Immunohistochemistry Following Spinal Cord Compression Trauma in the Rat and Mouse

Objective and methods To evaluate synaptic changes using synaptophysin immunohistochemstry in rat and mouse, which spinal cords were subjected to graded compression trauma at the level of Th8-9. Results Normal animals showed numerous fine dots of synaptophysin immunoreactivity in the gray matter. An increase in synaptophysin immunoreactivity was observed in the neuropil and synapses at the surface of motor neurons of the anterior horns in the ThS-9 segments lost immunoreactivity at 4-hour point after trauma. The immunoreactive synapses reappeared around motor neurons at 9-day point. Unexpected accumulation of synaptophysin immunoreactivity occurred in injured axons of the white matter of the compressed spinal cord. Conclusion Synaptic changes were important components of secondary injuries in spinal cord trauma. Loss of synapses on motor neurons may be one of the factors causing motor dysfunction of hind limbs and formation of new synapses may play an import,ant role in recovery of motor function. Synaptophysin immunohistochemistry is also a good tool for studies of axonal swellings in spinal cord injuries.

Gene therapy and the regeneration of retinal ganglion cell axons

Because the adult mammalian central nervous system （CNS） has only limited intrinsic capacity to regenerate connections after injury, due to factors both intrinsic and extrinsic to the mature neuron （Shen et al., 1999; Berry et al., 2008; Lingor et al., 2008; Sun and He, 2010; Moore et al., 2011 ）, therapies are required to support the survival of injured neu-rons and to promote the long-distance regrowth of axons back to their original target structures. The retina and optic nerve （ON） are part of the CNS and this system is much used in experiments designed to test new ways of promoting regeneration after injury （Harvey et al., 2006; Benowitz and Yin, 2008; Berry et al., 2008; Fischer and Leibinger, 2012）. Testing of therapies designed to improve retinal ganglion cell （RGC） viability also has direct clinical relevance because there is loss of these centrally projecting neurons in many ophthalmic diseases.

Physical interactions between activated microglia and injured axons:do all contacts lead to phagocytosis?

Axonal injury is a pathological hallmark of both head injury and inflammatory-mediated neurological disorders,including multiple sclerosis（Schirmer et al.,2013）.Such axonal disruptions and/or disconnections typically result in proximal axonal segments that remain in continuity with the neuronal somawhile losing contact with their distal targets.

Autophagy in degenerating axons following spinal cord injury: evidence for autophagosome biogenesis in retraction bulbs

Macroautophagy （here autophagy） is a catabolic mechanism responsible for the degradation of bulk cytoplasm, long-lived proteins and organeUes. During autophagy, the cargos are engulfed by double-membrane structures named phagophores, which expand to form the autophagosomes. Subsequently, these autophagosomes fuse with lysosomes, in which the cytoplasmic cargos are degraded. Autophagy is a constitutive pro- cess, which plays an important role in cellular homeostasis. In primary neurons autophagosome formation occurs continuously and preferentially at the distal end of axons. On the other hand, autophagy is increased by different stresses, and its dysregulation or excessive induction may lead to detrimental effects. Many neurological disorders have been associated with alterations in the autophagic pathway and an increase in autophagy during axonal degeneration was described.

Distinct effect of potassium 2-(l-hydroxypentyl) - benzoate on hippocampal neurons, synapses and dystrophic axons in APP/PS1 mice

OBJECTIVE To study the protective effect of potassium 2-(l-hydroxypentyl)-benzoate(PHPB) on hippocampal neurons,synapses and dystrophic axons in APP/PS1 mice.METHODS Ten-month-old male APP/PS1 transgenic mice and age-matched wild-type mice were randomly divided into three groups:wild-type group(WT Con group,n=10),APP/PS1 group(Tg Con group,n=10) and PHPB treated APP/PS1 group(PHPB group,n=10).PHPB group received 30 mg · kg-1 PHPB by oral gavage once daily for 3 months.WT Con group and Tg Con group received the same volume of water.Three months later,mice were sacrificed for biochemical and pathological testing such as transmission electron microscopy,Golgi staining and Western boltting analysis.RESULTS Under the transmission electron microscope,most hippocampal neurons and subcel ular organel es in WT Con group exhibited normal morphology.However,the degenerative changes were observed in Tg Con group such as nuclear fragmentation,mitochondrial swelling,ribosomes detachment and autophagic vacuoles accumulation.The hippocampal synapses number and the thickness of postsynaptic density(PSD) were significantly decreased in Tg Con group compared with the WT Con group(P<0.05).After PHPB treatment,the degenerative changes in APP/PS1 mice were alleviated to some extent.The synapse number has been elevated significantly(P<0.05) and the PSD has been thickened as well.Golgi staining showed that the spine density of secondary and tertiary apical dendritic branches was significantly decreased in CA1 and DG areas of Tg Con group(P<0.05).Sholl analysis revealed a decrease of dendritic complexity in Tg Con group compared with WT Con group(P<0.05).These abnormalities were alleviated to some extent after PHPB treatment.Western blotting study showed that the protein levels of synaptic marker PSD-95 and synaptophysin were significantly decreased in the hippocampus of Tg Con group(P<0.05).A significant increase of PSD-95(P<0.05) and a slight increase of SYP were observed after the PHPB treatment.Besides,we found a significant increase in the ratio of LC3-Ⅱ/LC3-Ⅰ in Tg Con group compared with the WT Con group(P<0.01) and the relevant improvement after PHPB treatment(P<0.05),which showed the regulatory effect of PHPB on autophagy impairment.CONCLUSION PHPB showed protective effects on hippocampal neurons,synapses and dystrophic axons in APP/PS1 mice,which might help explain its role on cognitive improvement in Alzheimer disease treatment.

Reassembly of the axon initial segment and nodes of Ranvier in regenerated axons of the central nervous system

Myelinated axons of the peripheral and central nervous system（PNS＆CNS）are divided into molecularly distinct excitable domains,including the axon initial segment（AIS）and nodes of Ranvier.The AIS is composed of a dense network of cytoskeletal proteins,cell adhesion molecules,and voltage gated ion channels and is located at the proximal most region of the axon（Koleand Stuart, 2012）.

Long range electromagnetic field nature of nerve signal propagation in myelinated axons

The nature of saltatory conduction in myelinated axon described by equivalent circuit and circuit theory is still contentious. Recent experimental observations of action potentials transmitting through disjointed nerve fibers strongly suggest an electromagnetic wave propagation mechanism of the nerve signals. In this paper, we employ the electromagnetic wave model of the myelinated axon to describe action potential signal propagation. We use the experimental frequency-dependent conductivity and permittivity values of the nerve tissues in order to reliably calculate the electromagnetic modes by using electromagnetic mode solvers. We find that the electromagnetic waves above 10 kHz can be well confined in extracellular fluid–myelin sheath–intracellular fluid waveguide and propagate a distance of 7 mm without much attenuation. Our study may serve as one of the fundamental researches for the better understanding of the nervous system.

CHARACTERIZATION OF SIGNAL CONDUCTIONALONG DEMYELINATED AXONS BY ACTION-POTENTIAL-ENCODED SECOND HARMONICGENERATION

Action-potential encoded optical second harmonic generation(SHG)has been recently proposedfor use in det ecting the axonal damage in patients with demnyelinat ing diseases.In this study,thecharact erization of signal conduction along axons of two different levels of denyelination wasstudied via a modified Hodgkin Huxley model,because some types of demyelinating disease,i.e.primary progressive and secondary progesive multiple scleross,are dificult to be distinguishedby magnetic resonance imaging(MRI),we focused on the diferences in signal conduction between two diferent demyelinated axons,such as the first-level demyelination and the second.level demyelination.The spatio-temporal distribution of action potentials along denyelinatedaxons and conduction properties including the refractory period and frequency encoding in theset wo patterns were investigated.The results showed that denyelination could induce the decreaseboth in the amplitude of action potentials and the ability of frequency coding,Furthermore,t hesignal conduction velocity in the second-level dernyelination was about 21%slower than that inthe first-level demyelination.The refractory period in the second-level demyelination was about32%longer t han the first-level.Thus,detecting the signal conduction in demnyelinat ed axons byaction-potential encoded optical SHG could greatly improve the assessment of demyelinatingdisorders to classify the patients.This technique also offers a potential fast and noninvasiveoptical approach for monitoring membrane potential.

Local translation of cell adhesion molecules in axons

During development and regeneration,axonal growth depends on a rapid response to extracellular growth and guidance molecules.One mechanism underlying this rapid response is local protein synthesis（Jung et al.,2012）.Local protein synthesis is a highly tuned,

Targeting transcriptional regulators to regenerate midbrain dopaminergic axons in Parkinson's disease

Introduction： Parkinson＇s disease （PD） is a chronic, age-re- lated neurodegenerative disorder that affects 1-2% of the population over the age of 65. PD is characterised by the progressive degeneration of nigrostriatal dopaminergic （DA） neurons. This leads to disabling motor symptoms, due to the striatal DA denervation. Despite decades of research, there is still no therapy that can slow, stop or regenerate the dying midbrain DA neurons in PD.

Retinal ganglion cells regenerate long-distance axons through neural activity stimulation and find their way back to the brain

Human central nerve system(CNS)is an extremely complex and delicate structure.While regeneration is possible in some reptiles and fish CNS,the regeneration capacity seems completely lost in adult mammals.Therefore,the classic concept is that once neurons in mammal

Radial glia interact with primary olfactory axons to regulate development of the olfactory bulb

The developing olfactory system - merging of the periph- eral and central nervous systems： The olfactory system is responsible for the sense of smell and is comprised of a complex topographic map that regenerates throughout life. In rodents each olfactory sensory neuron expresses one of N 1,300 odorant receptors with the neurons being distributed mosaically within the epithelium. The axons of the sensory neurons do not maintain near-neighbour relationships and instead project to disparate topographic targets in the olfac- tory bulb within the central nervous system. The develop- ment of the targets relies on the intermingling of the sensory axons with the interneurons, glia and second order neurons of the olfactory bulb. Thus the formation of the olfactory system involves the coordinated integration of the axons of the peripheral olfactory sensory neurons with the cells of the olfactory bulb.

Specific effects of c-Jun NH2-terminal kinaseinteracting protein 1 in neuronal axons

c-Jun NH2-terminal kinase（JNK）-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B（Trk B） anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of Trk B anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed Trk B complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of Trk B gradually increased in axon terminals. However, the distribution of Trk B reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of Trk B after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of Trk B in dendrites. These findings confirm that JNK-interacting protein 1 can interact with Trk B in neuronal cells, and can regulate the transport of Trk B in axons, but not in dendrites.

Structure and function of the contactin-associated protein family in myelinated axons and their relationship with nerve diseases

The contactin-associated protein （Caspr） family participates in nerve excitation and conduction, and neurotransmitter release in myelinated axons. We analyzed the structures and functions of the Caspr family- CNTNAP1 （Casprl）, CNTNAP2 （Caspr2）, CNTNAP3 （Caspr3）, CNTNAP4 （Caspr4） and CNTNAP5 （Caspr5）, Casprl-5 is not only involved in the formation of myelinated axons, but also participates in maintaining the stability of adjacent connections. Casprl participates in the formation, differentiation, and proliferation of neurons and astrocytes, and in motor control and cognitive function. We also analyzed the relationship between the Caspr family and neurodegenerative diseases, multiple sclerosis, and autoimmune encephalitis. However, the effects of Caspr on disease course and prognosis remain poorly understood. The effects of Caspr on disease diagnosis and treatment need further investigation.

Glial scar size, inhibitor concentration, and growth of regenerating axons after spinal cord transection

A mathematical model has been formulated in accordance with cell chemotaxis and relevant experimental data. A three-dimensional lattice Boltzmann method was used for numerical simulation. The present study observed the effects of glial scar size and inhibitor concentration on regenerative axonal growth following spinal cord transection. The simulation test comprised two parts： （1） when release rates of growth inhibitor and promoter were constant, the effects of glial scar size on axonal growth rate were analyzed, and concentrations of inhibitor and promoters located at the moving growth cones were recorded. （2） When the glial scar size was constant, the effects of inhibitor and promoter release rates on axonal growth rate were analyzed, and inhibitor and promoter concentrations at the moving growth cones were recorded. Results demonstrated that （1） a larger glial scar and a higher release rate of inhibitor resulted in a reduced axonal growth rate. （2） The axonal growth rate depended on the ratio of inhibitor to promoter concentrations at the growth cones. When the average ratio was 〈 1.5, regenerating axons were able to grow and successfully contact target cells.

Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons

Most current studies quantify axon regeneration by immunostaining regeneration-associated proteins,representing indirect measurement of axon lengths from both sensory neurons in the dorsal root ganglia and motor neurons in the spinal cord.Our recently developed method of in vivo electroporation of plasmid DNA encoding for enhanced green fluorescent protein into adult sensory neurons in the dorsal root ganglia provides a way to directly and specifically measure regenerating sensory axon lengths in whole-mount nerves.A mouse model of sciatic nerve compression was established by squeezing the sciatic nerve with tweezers.Plasmid DNA carrying enhanced green fluorescent protein was transfected by ipsilateral dorsal root ganglion electroporation 2 or 3 days before injury.Fluorescence distribution of dorsal root or sciatic nerve was observed by confocal microscopy.At 12 and 18 hours,and 1,2,3,4,5,and 6 days of injury,lengths of regenerated axons after sciatic nerve compression were measured using green fluorescence images.Apoptosis-related protein caspase-3 expression in dorsal root ganglia was determined by western blot assay.We found that in vivo electroporation did not affect caspase-3 expression in dorsal root ganglia.Dorsal root ganglia and sciatic nerves were successfully removed and subjected to a rapid tissue clearing technique.Neuronal soma in dorsal root ganglia expressing enhanced green fluorescent protein or fluorescent dye-labeled microRNAs were imaged after tissue clearing.The results facilitate direct time course analysis of peripheral nerve axon regeneration.This study was approved by the Institutional Animal Care and Use Committee of Guilin Medical University,China(approval No.GLMC201503010)on March 7,2014.

The functional properties of synapses made by regenerated axons across spinal cord lesion sites in lamprey

While the anatomical properties of regenerated axons across spinal cord lesion sites have been studied extensively,little is known of how the functional properties of regenerated synapses compared to those in unlesioned animals.This study aims to compare the properties of synapses made by regenerated axons with unlesioned axons using the lamprey,a model system for spinal injury research,in which functional locomotor recovery after spinal cord lesions is associated with axonal regeneration across the lesion site.Regenerated synapses below the lesion site did not differ from synapses from unlesioned axons with respect to the amplitude and duration of single excitatory postsynaptic potentials.They also showed the same activity-dependent depression over spike trains.However,regenerated synapses did differ from unlesioned synapses as the estimated number of synaptic vesicles was greater and there was evidence for increased postsynaptic quantal amplitude.For axons above the lesion site,the amplitude and duration of single synaptic inputs also did not differ significantly from unlesioned animals.However,in this case,there was evidence of a reduction in release probability and inputs facilitated rather than depressed over spike trains.Synaptic inputs from single regenerated axons below the lesion site thus do not increase in amplitude to compensate for the reduced number of descending axons after functional recovery.However,the postsynaptic input was maintained at the unlesioned level using different synaptic properties.Conversely,the facilitation from the same initial amplitude above the lesion site made the synaptic input over spike trains functionally stronger.This may help to increase propriospinal activity across the lesion site to compensate for the lesion-induced reduction in supraspinal inputs.The animal experiments were approved by the Animal Ethics Committee of Cambridge University.