Injured Neurons Research Articles

Temporal gene expression changes after acute and delayed ventral root avulsion-reimplantation.

In a model of injured spinal motor neurons where the avulsed spinal nerve is surgically reimplanted, useful regrowth of the injured nerve follows, both in animal experiments and clinical cases. This has led to surgical reimplantation strategies with subsequent partial functional motoric recovery. Still, the ideal time point for successful regeneration after reimplantation and the specific genetic profile of this time point is not known. To explore the temporal gene expression of the whole genome in the ventral spinal cord after reimplantation at different time points after avulsion. Totally 18 adult rats were subjected to avulsion of the left L5 root only (N = 3), avulsion followed by acute spinal reimplantation (N = 3), avulsion followed by 24 h (N = 3) or 48 h (N = 3) delayed reimplantation. Animals were allowed to survive 24 h after their respective surgery whereafter the ventral quadrant of the spinal cord at the operated side was harvested, processed for and analysed with Affymetrix Rat Gene ST 1.0 array followed by statistical analysis of gene expression patternsResults:Specific gene expression patterns were found at different time points after avulsion and reimplantation. Over all, early reimplantation seemed to diminish inflammatory response and support gene regulation related to neuronal activity compared to avulsion only or delayed reimplantation. In addition did gene activity after avulsion-reimplantation correspond to regeneration-associated genes typical for regeneration in the peripheral nervous system. Our study reveal that genetic profiling after this kind of injury is possible, that specific and distinct expression patterns can be found with early reimplantation being favourable over late and that regenerative activity in this kind of injury bears hallmark typical for peripheral nerve regeneration. These findings can be useful in elucidating specific genetic expression typical for successful nerve regeneration, hopefully not only in this specific model but in the nervous system in general.

Role of neurotrophic factors in enhancing linear axonal growth of ganglionic sensory neurons in vitro.

Neurotrophins play a major role in the regulation of neuronal growth such as neurite sprouting or regeneration in response to nerve injuries. The role of nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor in maintaining the survival of peripheral neurons remains poorly understood. In regenerative medicine, different modalities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. This study was to investigate the influence of nerve growth factor, neurotrophin-3 and brain-derived neurotrophic factor on the growth of neurites using two in vitro models of dorsal root ganglia explants and dorsal root ganglia-derived primary cell dissociated cultures. Quantitative data showed that the total neurite length and tortuosity were differently influenced by trophic factors. Nerve growth factor and, indirectly, brain-derived neurotrophic factor stimulate the tortuous growth of sensory fibers and the formation of cell clusters. Neurotrophin-3, however, enhances neurite growth in terms of length and linearity allowing for a more organized and directed axonal elongation towards a peripheral target compared to the other growth factors. These findings could be of considerable importance for any clinical application of neurotrophic factors in peripheral nerve regeneration. Ethical approval was obtained from the Regione Piemonte Animal Ethics Committee ASLTO1 (file # 864/2016-PR) on September 14, 2016.

Astrocytes-derived exosomes induce neuronal recovery after traumatic brain injury via delivering gap junction alpha 1-20 k.

Astrocytes are more resistant to ischemia and hypoxia in the acute phase of brain injury after traumatic brain injury (TBI). Previous study showed that gap junction alpha 1 (GJA1) phosphorylation can increase the survival of damaged astrocytes. The GJA1-20 k expression in neurons co-culture with astrocytes was positively correlated with exosomes uptake. This study aims to explore the effect of exogenous GJA1-20 k carried by astrocyte-derived exosomes on neurons apoptosis and mitochondrial function after TBI. Astrocytes were co-cultured with the neuron with/without damage from air pressure. Exosomes were isolated, extracted from the culture medium by differential ultra-centrifugation, and verified by electron microscopy. Immunofluorescence staining, tunnel, western blot were employed to detect exosomes marker CD60, apoptosis, and mitochondrial function related protein expression and GJA1-20 k in cell culture. A rat model of hydraulic injury TBI was built, and exosomes was transferred. 2,3,5-Triphenyltetrazolium chloride (TTC) staining and immunohistochemistry staining of Nissl and microtubule associated protein 2 were used to detect the brain damage. A transwell stereo culture model of astrocytes and TBI-like injured neuron was constructed. The exosomes derived from astrocytes promoted the recovery of damaged neuron by in vitro exosome treatment. Compared with GJA1-20 k knockout exosome control group, GJA1-20 k exosomes were uptaken by neuron and downregulated the apoptosis rate and upregulated mitochondrial function to promote neuronal recovery. Finally, the results were validated by TTC staining and damaged tissue sections of rat TBI model. This study contributes to a better understanding of the astrocyte-neuron protection mechanism in TBI and provides a potential new target for the treatment of TBI.

Mesenchymal Stem Cell-Derived Exosomes and Other Extracellular Vesicles as New Remedies in the Therapy of Inflammatory Diseases.

There is growing evidence that mesenchymal stem cell (MSC)-based immunosuppression was mainly attributed to the effects of MSC-derived extracellular vesicles (MSC-EVs). MSC-EVs are enriched with MSC-sourced bioactive molecules (messenger RNA (mRNA), microRNAs (miRNAs), cytokines, chemokines, immunomodulatory factors) that regulate phenotype, function and homing of immune cells. In this review article we emphasized current knowledge regarding molecular mechanisms responsible for the therapeutic effects of MSC-EVs in attenuation of autoimmune and inflammatory diseases. We described the disease-specific cellular targets of MSC-EVs and defined MSC-sourced molecules, which were responsible for MSC-EV-based immunosuppression. Results obtained in a large number of experimental studies revealed that both local and systemic administration of MSC-EVs efficiently suppressed detrimental immune response in inflamed tissues and promoted survival and regeneration of injured parenchymal cells. MSC-EVs-based anti-inflammatory effects were relied on the delivery of immunoregulatory miRNAs and immunomodulatory proteins in inflammatory immune cells (M1 macrophages, dendritic cells (DCs), CD4+Th1 and Th17 cells), enabling their phenotypic conversion into immunosuppressive M2 macrophages, tolerogenic DCs and T regulatory cells. Additionally, through the delivery of mRNAs and miRNAs, MSC-EVs activated autophagy and/or inhibited apoptosis, necrosis and oxidative stress in injured hepatocytes, neurons, retinal cells, lung, gut and renal epithelial cells, promoting their survival and regeneration.

Determining Neurotrophin Gradients in Vitro To Direct Axonal Outgrowth Following Spinal Cord Injury.

A spinal cord injury can damage neuronal connections required for both motor and sensory function. Barriers to regeneration within the central nervous system, including an absence of neurotrophic stimulation, impair the ability of injured neurons to reestablish their original circuitry. Exogenous neurotrophin administration has been shown to promote axonal regeneration and outgrowth following injury. The neurotrophins possess chemotrophic properties that guide axons toward the region of highest concentration. These growth factors have demonstrated potential to be used as a therapeutic intervention for orienting axonal growth beyond the injury lesion, toward denervated targets. However, the success of this approach is dependent on the appropriate spatiotemporal distribution of these molecules to ensure detection and navigation by the axonal growth cone. A number of in vitro gradient-based assays have been employed to investigate axonal response to neurotrophic gradients. Such platforms have helped elucidate the potential of applying a concentration gradient of neurotrophins to promote directed axonal regeneration toward a functionally significant target. Here, we review these techniques and the principles of gradient detection in axonal guidance, with particular focus on the use of neurotrophins to orient the trajectory of regenerating axons.

Gabapentinoid treatment promotes corticospinal plasticity and regeneration following murine spinal cord injury.

Axon regeneration failure causes neurological deficits and long-term disability after spinal cord injury (SCI). Here, we found that the α2δ2 subunit of voltage-gated calcium channels negatively regulates axon growth and regeneration of corticospinal neurons, the cells that originate the corticospinal tract. Increased α2δ2 expression in corticospinal neurons contributed to loss of corticospinal regrowth ability during postnatal development and after SCI. In contrast, α2δ2 pharmacological blockade through gabapentin administration promoted corticospinal structural plasticity and regeneration in adulthood. Using an optogenetic strategy combined with in vivo electrophysiological recording, we demonstrated that regenerating corticospinal axons functionally integrate into spinal circuits. Mice administered gabapentin recovered upper extremity function after cervical SCI. Importantly, such recovery relies on reorganization of the corticospinal pathway, as chemogenetic silencing of injured corticospinal neurons transiently abrogated recovery. Thus, targeting α2δ2 with a clinically relevant treatment strategy aids repair of motor circuits after SCI.

Hypoglossal nerve injury with long nerve resection leading to slow motoneuron death

Crush injury to peripheral nerves in adult animals is considered not to trigger retrograde neuronal cell death; however, several studies reported neuronal cell death following severe injuries including nerve transection, resection, or avulsion. However, the rate of neuronal cell death varied among studies. In this study, we evaluated the outcomes of very severe nerve injury by long nerve resection in adult rats. Right hypoglossal (XII) nerve was exposed, and a 9-mm section was resected. At 4, 8, and 12 weeks after the resection, the number of XII neurons were counted in from the rostral to caudal sections. The number of XII neurons in the injured right side was reduced after the XII nerve resection compared with the uninjured left side. The mean rates of surviving neurons at 4, 8, and 12 weeks after the nerve resection were 83.5 %, 73.9 %, and 61.1 %, respectively, which were significantly lower than those of the control. The number of XII neurons after extensive XII nerve resection declined gradually over a relatively long time period, revealing that extensive nerve resection led to slow cell death of the injured neurons.

Mesenchymal stem cells in chemotherapy-induced peripheral neuropathy: A new challenging approach that requires further investigations.

Chemotherapeutic drugs may disrupt the nervous system and cause chemotherapy-induced peripheral neuropathy (CIPN) as side effects. There are no completely successful medications for the prevention or treatment of CIPN. Many drugs such as tricyclic antidepressants and anticonvulsants have been used for symptomatic treatment of CIPN. Unfortunately, these drugs often give only partial relief or have dose-limiting side effects. Thus, the treatment of CIPN becomes a challenge because of failure to regenerate and repair the injured neurons. Mesenchymal stem cell (MSC) therapy is a new attractive approach for CIPN. Evidence has demonstrated that MSCs play important roles in reducing oxidative stress, neuroinflammation, and apoptosis, as well as mediating axon regeneration after nerve damage in several experimental studies and some clinical trials. We will briefly review the pathogenesis of CIPN, traditional therapies used and their drawbacks as well as therapeutic effects of MSCs, their related mechanisms, future challenges for their clinical application, and the additional benefit of their combination with pharmacological agents. MSCs-based therapies may provide a new therapeutic strategy for patients suffering from CIPN where further investigations are required for studying their exact mechanisms. Combined therapy with pharmacological agents can provide another promising option for enhancing MSC therapy success while limiting its adverse effects.

GSK3/CRMP2 signaling confers PTEN knockout mediated axonal regeneration

AbstractKnockout of phosphatase and tensin homolog (PTEN‐/‐) is neuroprotective and promotes axon regeneration in mature neurons. Elevation and maintenance of mTOR activity in injured neurons have been proposed as the main underlying mechanism. Here we demonstrate that PTEN‐/‐ also abrogates the inhibitory activity of GSK3 on collapsin response mediator protein 2 (CRMP2) in retinal ganglion cell (RGC) axons. Moreover, maintenance of GSK3 activity in GSK3S/A knockin mice significantly compromised PTEN‐/‐‐mediated optic nerve regeneration as well as the activity of CRMP2, and to a lesser extent, mTOR. These GSK3S/A mediated negative effects on regeneration were rescued by viral expression of constitutively active CRMP2T/A, despite decreased mTOR activation. GSK3S/A knockin or CRMP2 inhibition also decreased PTEN‐/‐ mediated neurite growth of RGCs in culture and disinhibition towards CNS myelin. Thus, the GSK3/CRMP2 pathway is essential for PTEN‐/‐ mediated axon regeneration. These new mechanistic insights may help to find novel strategies to promote axon regeneration

The effect of peri-operative hyperbaric oxygen therapy on the recovery of neurologic function of patients with cervical spondylotic myelopathy: A diffusion tensor imaging study

Objective To observe the clinical effect of hyperbaric oxygen therapy on cervical spondylotic myelopathy (CSM) in the peri-operative period, and to explore its neural mechanism. Methods Eighty patients who underwent surgical decompression for CSM were randomly divided into a hyperbaric oxygen group (n=40) and a control group (n=40). Both groups received cervical laminoplasty and systematic rehabilitation treatment after the surgery, while the hyperbaric oxygen group was additionally provided with hyperbaric oxygen therapy in the peri-operative period. The patients′ neurological status was evaluated using Japanese Orthopaedic Association (JOA) scores. Both groups received conventional MRI and diffusion tensor imaging (DTI) before and 6 months after the surgery. Results After the surgery, both groups gained significant improvement in their average JOA score, with the improvement of the hyperbaric oxygen group significantly greater than in the control group. Pearson correlation analysis showed that the average pre-operative JOA score was significantly correlated with the anisotropic fraction (FA) and the apparent dispersion coefficient (ADC) of the compressed spinal cord. Six months after the surgery such correlation still persisted. During the six months, significant increase was observed in the average FA and significant decrease in the average ADC in both groups, with the average FA of the hyperbaric oxygen group (0.726±0.087) significantly higher at the end of the 6 months than that in the control group. The average ADC (1.148±0.079)×10-3 mm2/s) was significantly lower. Conclusions DTI′s quantitative indicators can objectively show changes in the microstructure and pathological state of spinal cords. Exposure to hyperbaric oxygen may relieve ischemia and hypoxia of the spinal cord, promoting the repair of injured neurons and accelerating the regeneration of nerve fibers. Key words: Hyperbaric oxygen; Cervical spine; Spondylosis; Myelopathy; Laminoplasty; Diffusion tensor imaging

ERRγ is downregulated in injured motor neuron subpopulations following brachial plexus root avulsion.

Estrogen-related receptor γ (ERRγ) is a member of a small group of orphan nuclear receptor transcription factors that have been implicated in several physiological and pathological processes, including placental development, regulation of metabolic genes or disease. The pattern of expression of ERRγ, its role in neuronal injury and its co-localization with other transcription factors in the spinal cord of rats with brachial plexus injury has not been determined. The expression profile of ERRγ and its co-localization with RNA binding protein fox-1 homolog 3 (NeuN) or cyclic AMP-dependent transcription factor 3 (ATF-3) in the motor neurons of rats that underwent brachial plexus root avulsion were assessed using western blot analysis, immunohistochemistry and immunofluorescence. Fluorogold (FG) was used to mark neurons whose axons were severed. ATF-3 was expressed in the nuclei of motor neurons whose axons were severed by root avulsion. On day 3 post-avulsion, FG and ATF-3 were all co-localized in the injured motor neurons. The level of ERRγ protein in the ipsilateral half of injured spinal cords was significantly decreased compared with that in the contralateral half on days 3, 14 and 28 post-avulsion (all P<0.05). The numbers of ERRγ-positive motor neurons (ERRγon) were also notably decreased in the ipsilateral side compared with that in the contralateral side on days 14 and 28 post-avulsion, implying that the expression occurred in α motor neurons that were progressively being lost, a phenomenon that was expected post-brachial plexus avulsion. Almost all large and small ERRγ-positive motor neurons were also NeuN-positive (NeuNon). However, a few of these were ERRγon/NeuNoff (no NeuN signal). Therefore, these results suggested that ERRγ is a non-specific marker of γ motor neurons in rats, and therefore, this specific transcriptional program cannot be used to define functionally distinct motor neuron sub-populations. However, its downregulation on the injured side suggests that it is an important component of the response to injury in motor neurons.

MES23.5 DA Immortalized Neuroblastoma Cells Self-protect Against Early Injury by Overexpressing Glial Cell-derived Neurotrophic Factor via Akt1/Eya1/Six2 Signaling.

Injured neurons can initiate their own neurotoxin-induced repair mechanisms by expressing protective genes and activating specific intracellular signal transduction pathways. Although glial cell-derived neurotrophic factor (GDNF) plays a key role in the repair of dopaminergic (DA) neurons, whether there is high expression of GDNF in DA neurons at an early stage of injury has not yet been reported. In this study, neurotoxin-induced GDNF overexpression was detected for the first time in MES23.5 DA immortalized neuroblastoma (MES23.5 DA) cells soon after 6-hydroxydopamine (6-OHDA) treatment. We also observed that the phosphorylation of Akt1, a member of the protein kinase B family, was increased. Further studies showed that activated Akt1 increased the phosphorylation of the protein phosphatase Eya1, which is a member of the eyes absent (Eya) family of transcriptional cofactors. Then, activated Eya1 decreased the phosphorylation of the sine oculis-related homeobox 2 (Six2) transcription factor. In addition, chromatin immunoprecipitation coupled with quantitative polymerase chain reaction (ChIP-qPCR) revealed that Six2 promoted GDNF transcription in MES23.5 DA cells by directly binding to the GDNF promoter. Finally, we showed that inhibiting neurotoxin-induced GDNF overexpression increased MES23.5 DA cell death, while promoting GDNF expression via Six2 overexpression decreased DA neuronal death. These results suggest that MES23.5 DA cells with early 6-OHDA-induced injury can promote the overexpression of GDNF by activating the Akt1/Eya1/Six2 signaling pathway, and this overexpression of GDNF has protective effects on injured MES23.5 DA cells. Hence, this study highlights a new target for drug development for the treatment of Parkinson's disease.

Automated label-free detection of injured neuron with deep learning by two-photon microscopy.

Stroke is a significant cause of morbidity and long-term disability globally. Detection of injured neuron is a prerequisite for defining the degree of focal ischemic brain injury, which can be used to guide further therapy. Here, we demonstrate the capability of two-photon microscopy (TPM) to label-freely identify injured neurons on unstained thin section and fresh tissue of rat cerebral ischemia-reperfusion model, revealing definite diagnostic features compared with conventional staining images. Moreover, a deep learning model based on convolutional neural network is developed to automatically detect the location of injured neurons on TPM images. We then apply deep learning-assisted TPM to evaluate the ischemic regions based on tissue edema, two-photon excited fluorescence signal intensity, as well as neuronal injury, presenting a novel manner for identifying the infarct core, peri-infarct area, and remote area. These results propose an automated and label-free method that could provide supplementary information to augment the diagnostic accuracy, as well as hold the potential to be used as an intravital diagnostic tool for evaluating the effectiveness of drug interventions and predicting potential therapeutics.

Invertebrate Retinal Progenitors as Regenerative Models in a Microfluidic System.

Regenerative retinal therapies have introduced progenitor cells to replace dysfunctional or injured neurons and regain visual function. While contemporary cell replacement therapies have delivered retinal progenitor cells (RPCs) within customized biomaterials to promote viability and enable transplantation, outcomes have been severely limited by the misdirected and/or insufficient migration of transplanted cells. RPCs must achieve appropriate spatial and functional positioning in host retina, collectively, to restore vision, whereas movement of clustered cells differs substantially from the single cell migration studied in classical chemotaxis models. Defining how RPCs interact with each other, neighboring cell types and surrounding extracellular matrixes are critical to our understanding of retinogenesis and the development of effective, cell-based approaches to retinal replacement. The current article describes a new bio-engineering approach to investigate the migratory responses of innate collections of RPCs upon extracellular substrates by combining microfluidics with the well-established invertebrate model of Drosophila melanogaster. Experiments utilized microfluidics to investigate how the composition, size, and adhesion of RPC clusters on defined extracellular substrates affected migration to exogenous chemotactic signaling. Results demonstrated that retinal cluster size and composition influenced RPC clustering upon extracellular substrates of concanavalin (Con-A), Laminin (LM), and poly-L-lysine (PLL), and that RPC cluster size greatly altered collective migratory responses to signaling from Fibroblast Growth Factor (FGF), a primary chemotactic agent in Drosophila. These results highlight the significance of examining collective cell-biomaterial interactions on bio-substrates of emerging biomaterials to aid directional migration of transplanted cells. Our approach further introduces the benefits of pairing genetically controlled models with experimentally controlled microenvironments to advance cell replacement therapies.

Stereotyped transcriptomic transformation of somatosensory neurons in response to injury.

In mice, spared nerve injury replicates symptoms of human neuropathic pain and induces upregulation of many genes in somatosensory neurons. Here we used single cell transcriptomics to probe the effects of partial infraorbital transection of the trigeminal nerve at the cellular level. Uninjured neurons were unaffected by transection of major nerve branches, segregating into many different classes. In marked contrast, axotomy rapidly transformed damaged neurons into just two new and closely-related classes where almost all original identity was lost. Remarkably, sensory neurons also adopted this transcriptomic state following various minor peripheral injuries. By genetically marking injured neurons, we showed that the injury-induced transformation was reversible, with damaged cells slowly reacquiring normal gene expression profiles. Thus, our data expose transcriptomic plasticity, previously thought of as a driver of chronic pain, as a programed response to many types of injury and a potential mechanism for regulating sensation during wound healing.

The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells.

Slit1 is one of the known signaling factors of the slit family and can promote neurite growth by binding to its receptor, Robo2. Upregulation of Slit1 expression in dorsal root ganglia (DRG) after peripheral nerve injury plays an important role in nerve regeneration. Each sensory neuronal soma in the DRG is encapsulated by several surrounding satellite glial cells (SGCs) to form a neural structural unit. However, the temporal and spatial patterns of Slit1 upregulation in SGCs in DRG and its molecular mechanisms are not well understood. This study examined the spatial and temporal patterns of Slit1 expression in DRG after sciatic nerve crush by immunohistochemistry and western blotting. The effect of neuronal damage signaling on the expression of Slit1 in SGCs was studied in vivo by fluorescent gold retrograde tracing and double immunofluorescence staining. The relationship between the expression of Slit1 in SGCs and neuronal somas was also observed by culturing DRG cells and double immunofluorescence labeling. The molecular mechanism of Slit1 was further explored by immunohistochemistry and western blotting after intraperitoneal injection of Bright Blue G (BBG, P2X7R inhibitor). The results showed that after peripheral nerve injury, the expression of Slit1 in the neurons and SGCs of DRG increased. The expression of Slit1 was presented with a time lag in SGCs than in neurons. The expression of Slit1 in SGCs was induced by contact with surrounding neuronal somas. Through injured cell localization, it was found that the expression of Slit1 was stronger in SGCs surrounding injured neurons than in SGCs surrounding non-injured neurons. The expression of vesicular nucleotide transporter (VNUT) in DRG neurons was increased by injury signaling. After the inhibition of P2X7R, the expression of Slit1 in SGCs was downregulated, and the expression of VNUT in DRG neurons was upregulated. These results indicate that the ATP-P2X7R pathway is involved in signal transduction from peripheral nerve injury to SGCs, leading to the upregulation of Slit1 expression.

The neuroprotective effect of mesenchymal stem cells is mediated through inhibition of apoptosis in hypoxic ischemic injury.

Neonatal hypoxia ischemia causes severe brain damage. Stem cell therapy is a promising method for treating neuronal diseases. Clinical translation of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) for the recovery of neurons after hypoxic ischemic encephalopathy (HIE) may represent an effective therapy. Primary neurons were exposed to oxygen-glucose deprivation (OGD) and subsequently cocultured with UC-MSCs. Apoptosis was examined by Annexin V-FITC-PI. Genes related to apoptosis were detected using RT-PCR and western-blot analyses. Using an in vivo model, HIE was induced in postnatal day 7 mice, and UC-MSCs were transplanted via the intraventricular route. UC-MSC migration was investigated by immunofluorescence, and lesion volumes were measured by TTC staining. Apoptosis in injured brain cells was detected by the TUNEL assay. RT-PCR and ELISA were used to detect the expression of inflammatory factors in cells and animal tissues. Flow cytometry analysis revealed that apoptosis in injured neurons was inhibited by UC-MSCs. The RT-PCR and western blot results indicated that coculture inhibited the expression of proapoptotic genes and upregulated expression of antiapoptotic genes. In the animal model, transplanted UC-MSCs migrated toward the cerebral lesion site and decreased the lesion extent in HIE. TUNEL staining showed that the MSC group exhibited significantly reduced numbers of TUNEL-positive cells. RT-PCR and ELISA showed that UC-MSCs inhibited the upregulation of TNF-α and IL-1β in response to hypoxic ischemic injury. These results indicate that UC-MSCs exert neuroprotective effects against hypoxic ischemic injury by inhibiting apoptosis, and the mechanism appears to be through alleviating the inflammatory response.

CED-4 CARD domain residues can modulate non-apoptotic neuronal regeneration functions independently from apoptosis

A major challenge in regenerative medicine is the repair of injured neurons. Regeneration of laser-cut C. elegans neurons requires early action of core apoptosis activator CED-4/Apaf1 and CED-3/caspase. While testing models for CED-4 as a candidate calcium-sensitive activator of repair, we unexpectedly discovered that amino acid substitutions affecting alpha-helix-6 within the CED-4 caspase recruitment domain (CARD) confer a CED-4 gain-of-function (gf) activity that increases axonal regrowth without disrupting CED-4 apoptosis activity. The in vivo caspase reporter CA-GFP reveals a rapid localized increase in caspase activity upon axotomy, which is absent in ced-4 and ced-3 loss-of-function mutants but present in the ced-4(gf) mutant. The ced-3 loss-of-function mutation can significantly suppress the axonal regrowth of the ced-4(gf) mutant, indicating that CED-4(gf) regeneration depends on CED-3 caspase. Thus, we identified a subdomain within the CED-4 CARD that regulates the dynamic and controlled caspase activity required for efficient regeneration.

The Neuroprotective Effect of the HDAC2/3 Inhibitor MI192 on the Penumbra After Photothrombotic Stroke in the Mouse Brain.

Unilateral photothrombotic stroke caused tissue infarct in the mouse cerebral cortex. The injury of the cerebral cortex impaired the mouse motor activity, in particular the functional asymmetry in forelimb use. In the peri-infarct cortical tissue outside the infarctcore cell apoptosis occurred at 4 and 7days after PTS. The downregulation of acetylated α-tubulin, a marker of stable microtubules, showed the destruction of neurites, axons, and dendrites in injured neurons. However, the upregulation of GAP43 indicates the stimulation of neurite growth that was possibly aimed at the recovery of the cortical tissue in the damaged cerebral hemisphere. Application of MI-192, an inhibitor of histone deacetylases HDAC2 and HDAC3, demonstrated the neuroprotective activity in the mouse brain subjected to photothrombotic stroke. It reduced the volume of the PTS-induced infarction core in the mouse brain, partly restored the functional symmetry in the forelimb use, decreased the level of PTS-induced apoptosis and acetylation of α-tubulin characteristic for stable microtubules, and increased the expression of GAP-43 in the cerebral cortex of the damaged hemisphere. These data point to the involvement of HDAC2 and HDAC3 in the photothrombotic injury of the mouse brain not only in the infarction core but also outside it. The application of MI192 after PTS reduced or eliminated these negative effects and exerted the neuroprotective effect on the mouse brain. It may be a promising neuroprotector agent for anti-stroke therapy.

MC4R Is Involved in Neuropathic Pain by Regulating JNK Signaling Pathway After Chronic Constriction Injury.

BackgroundNeuropathic pain can develop after nerve injury, when deleterious changes occur in injured neurons and glia cells. Melanocortin 4 receptor (MC4R) is involved in the regulation of pain due to its high expressions in brain. Moreover, MC4R could mediate the c-Jun N-terminal kinase (JNK) signaling pathway, but whether the MC4R-regulated JNK signaling pathway participated in neuropathic pain after chronic constriction injury (CCI) is still unclear.MethodsA total of 128 Sprague-Dawley rats were allocated into four experiment groups: the SHAM group, CCI + NaCl group, CCI + HS group, and CCI + SP + HS group. For the CCI + NaCl group, the sciatic nerves were ligated. For the SHAM group, an identical manner to the CCI without ligation was performed. For CCI + HS and CCI + SP + HS groups, rats were injected with MC4R inhibitor (HS014) and HS014 plus JNK inhibitor (SP600125), respectively, from days 3 to 14 after CCI. Paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were used to assess the nociceptive behavior. ELISA was used to detect the levels of inflammatory cytokines. qRT-PCR and Western blots (WB) were utilized to examine the mRNA and protein expressions of JNK signaling pathway-related genes. Meanwhile, the expression levels of MC4R and p-JNK were further evaluated by immunohistochemistry (IHC) and immunofluorescence (IF) experiments. Finally, in order to confirm the in vivo results, astrocytes were isolated and transfected with MC4R-overexpression plasmid. Furthermore, the protein expressions of JNK signaling pathway-related genes were tested by WB.ResultsIt was showed that the values of PWL and PWT were significantly increased in CCI + HS group and CCI + SP + HS group compared with CCI + NaCl group. The increased interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) secretion in CCI + NaCl group was lowered by HS and SP + HS. MC4R, p-JNK, ATF3, and c-Jun levels were up-regulated with CCI surgery, but down-regulated with HS and SP + HS treatments. Moreover, the IHC and IF results further revealed that MC4R and p-JNK expressions in CCI + NaCl group were remarkably higher than those in HS group and HS + SP group. In vitro data also indicated that HS, SP, and SP + HS could down-regulate the expressions of MC4R, p-JNK, ATF3, and c-Jun in M1830 astrocytes.ConclusionOur findings indicated that MC4R is involved in neuropathic pain by regulating JNK signaling pathway after CCI.