RhoA as a target to promote neuronal survival and axon regeneration

Paralysis following spinal cord injury （SCI） is due to failure of axonal regeneration. It is believed that the capacities of neurons to regrow their axons are due partly to their intrinsic characteristics, which in turn are greatly influenced by several types of inhibitory molecules that are present, or even increased in the extracellular environment of the injured spinal cord. Many of these inhibitory molecules have been studied extensively in recent years. It has been suggested that the small GTPase RhoA is an intracellular convergence point for signaling by these extracellular inhibitory molecules, but due to the complexity of the central nervous system （CNS） in mammals, and the limitation of pharmacological tools, the specific roles of RhoA are unclear. By exploiting the anatomical and technical advantages of the lamprey CNS, we recently demonstrated that RhoA knockdown promotes true axon regeneration through the lesion site after SCI. In addition, we found that RhoA knockdown protects the large, identified reticulospinal neurons from apoptosis after their axons were axotomized in spinal cord. Therefore, manipulation of the RhoA signaling pathway may be an important approach in the development of treatments that are both neuroprotective and axon regeneration-promoting, to enhance functional recovery after SCI.

Nanomolar concentration of alpha-synuclein enhances dopaminergic neuronal survival via Akt pathway

Although alpha-synuclein is generally thought to have a pathological role in Parkinson＇s disease, accumulative evidence exists that alpha-synuclein has a neuroprotective effect. The aim of this study was to evaluate the effect of extracellular alpha-synuclein on dopaminergic cell survival. We assessed cell viability using the 3-（4,5-dimethyt-thiazol-2-yt）-2,5-diphenyltertazolium bromide （MTT） assay both in undifferentiated SH-SY5Y （SHSY） cells and neuronally-differentiated SH-SY5Y （ndSHSY） cells after 24 hour treatment with monomeric alpha-synuclein at various concentrations （0 [control], 50, 100 nmol/L, 1 IJmol/L）. To determine whether cell viability assessed by MTT assay was affected by cell proliferation, 5-bromo-2＇-deoxyuridine （BrdU） incorporation assay was per- formed. Level of both Akt and phosphorylated Akt was measured using western blot method in ndSHSY cells with or without 24 hour alpha-synuclein treatment. Cell viability was increased in ndSHSY cells at the nanomolar concentration of alpha-synuclein, but not in SHSY cells. Proportion of BrdU-positive ndSHSY cells was decreased in alpha-synuclein-treated group compared with control group. Level of phosphorylated Akt in alpha-synuclein-treated group was higher compared with the control group. Our study shows that extracellular alpha-synuclein at nanomolar concentra- tion benefits dopaminergic cell survival via Akt pathway.

Effect of Interleukin-1Beta (IL-1β) on the Cortical Neurons Survival and Neurites Outgrowth

Insults to the brain are known to cause a myriad of downstream effects, including the release of cytokines by astrocytes and resultant reactive gliosis. The author has examined effect of cytokine IL-1β on the survival of cortical neurons using mouse astrocyte-neuron co-culture. Five groups were used. These were neurons alone (Group 1), neurons with added IL-1β (Group 2), neurons co-cultured with astrocytes (Group 3), neurons co-cultured with astrocytes that was pre-treated with IL-1β before co-culture (Group 4) and neurons co-cultured with astrocytes and IL-1β added (post-treated) (Group 5). In Group 1 only a few neurons grew and survived only for 5-6 days. In Group 2, it was observed that more neurons survived up to 11 days. Moreover, in Group 3, more neurons grew and survived up to 16-18 days. They had large cell bodies and many long neurites that formed anastomosing networks. In Group 4, few neurons survived up to 13 days, whereas in Group 5, the growth of neurons were affected but to a much lesser extent than Group 4 and survived up to 15 days. In addition, it was found that IL-1β stimulated the expression of glial fibrillary acidic protein (GFAP) by astrocytes. This study indicates that IL-1β affects the survival of cortical neurons and modulates the astrocytic support to neuronal survival and neurites outgrowth by acting directly on the astrocytes.

Netrin-1 signaling pathway mechanisms in neurodegenerative diseases

Netrin-1 and its receptors play crucial roles in inducing axonal growth and neuronal migration during neuronal development.Their profound impacts then extend into adulthood to encompass the maintenance of neuronal survival and synaptic function.Increasing amounts of evidence highlight several key points:(1)Diminished Netrin-1 levels exacerbate pathological progression in animal models of Alzheimer’s disease and Parkinson’s disease,and potentially,similar alterations occur in humans.(2)Genetic mutations of Netrin-1 receptors increase an individuals’susceptibility to neurodegenerative disorders.(3)Therapeutic approaches targeting Netrin-1 and its receptors offer the benefits of enhancing memory and motor function.(4)Netrin-1 and its receptors show genetic and epigenetic alterations in a variety of cancers.These findings provide compelling evidence that Netrin-1 and its receptors are crucial targets in neurodegenerative diseases.Through a comprehensive review of Netrin-1 signaling pathways,our objective is to uncover potential therapeutic avenues for neurodegenerative disorders.

Insights into spinal muscular atrophy from molecular biomarkers

Spinal muscular atrophy is a devastating motor neuron disease characterized by severe cases of fatal muscle weakness.It is one of the most common genetic causes of mortality among infants aged less than 2 years.Biomarker research is currently receiving more attention,and new candidate biomarkers are constantly being discovered.This review initially discusses the evaluation methods commonly used in clinical practice while briefly outlining their respective pros and cons.We also describe recent advancements in research and the clinical significance of molecular biomarkers for spinal muscular atrophy,which are classified as either specific or non-specific biomarkers.This review provides new insights into the pathogenesis of spinal muscular atrophy,the mechanism of biomarkers in response to drug-modified therapies,the selection of biomarker candidates,and would promote the development of future research.Furthermore,the successful utilization of biomarkers may facilitate the implementation of gene-targeting treatments for patients with spinal muscular atrophy.

Dose-dependent and combined effects of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine on the survival of retinal ganglion cells in adult hamsters

This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine （L-NA） on the survival of retinal ganglion cells （RGCs） at 1 and 2 weeks after unilateral optic nerve transection in adult hamsters. The left optic nerves of all animals were transected intraorbitally 1 mm from the optic disc and RGCs were retrogradely labeled with Fluorogold before they received different daily dosages of single MK-801 or L-NA as well as daily combinational treatments of these two chemicals. All experimental and control animals survived for 1 or 2 weeks after optic nerve transection. Our results revealed that the mean numbers of surviving RGCs increased and then decreased when the dosage of MK-801 （1.0, 3.0 and 4.5 mg/kg） and L-NA （1.5, 3.0, 4.5 and 6.0 mg/kg） increased at both 1 and 2 weeks survival time points. Daily combinational use of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA lead to a highest RGC number that was even higher than the sum of the RGC numbers in 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA subgroups at 2 weeks. These findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these chemicals possesses a potentiative and protective effect.

Neurotrophic fragments as therapeutic alternatives to ameliorate brain aging

Aging is a global phenomenon and a complex biological process of all living beings that introduces various changes.During this physiological process,the brain is the most affected organ due to changes in its structural and chemical functions,such as changes in plasticity and decrease in the number,diameter,length,and branching of dendrites and dendritic spines.Likewise,it presents a great reduction in volume resulting from the contraction of the gray matter.Consequently,aging can affect not only cognitive functions,including learning and memory,but also the quality of life of older people.As a result of the phenomena,various molecules with notable neuroprotective capacity have been proposed,which provide a therapeutic alternative for people under conditions of aging or some neurodegenerative diseases.It is important to indicate that in recent years the use of molecules with neurotrophic activity has shown interesting results when evaluated in in vivo models.This review aims to describe the neurotrophic potential of molecules such as resveratrol(3,5,4′-trihydroxystilbene),neurotrophins(brain-derived neurotrophic factor),and neurotrophic-type compounds such as the terminal carboxyl domain of the heavy chain of tetanus toxin,cerebrolysin,neuropeptide-12,and rapamycin.Most of these molecules have been evaluated by our research group.Studies suggest that these molecules exert an important therapeutic potential,restoring brain function in aging conditions or models of neurodegenerative diseases.Hence,our interest is in describing the current scientific evidence that supports the therapeutic potential of these molecules with active neurotrophic.

Stimulating effect of thyroid hormones in peripheral nerve regeneration:research history and future direction toward clinical therapy

Injury to peripheral nerves is often observed in the clinic and severe injuries may cause loss of motor and sensory functions.Despite extensive investigation,testing various surgical repair techniques and neurotrophic molecules,at present,a satisfactory method to ensuring successful recovery does not exist.For successful molecular therapy in nerve regeneration,it is essential to improve the intrinsic ability of neurons to survive and to increase the speed of axonal outgrowth.Also to induce Schwann cell phenotypical changes to prepare the local environment favorable for axonal regeneration and myelination.Therefore,any molecule that regulates gene expression of both neurons and Schwann cells could play a crucial role in peripheral nerve regeneration.Clinical and experimental studies have reported that thyroid hormones are essential for the normal development and function of the nervous system,so they could be candidates for nervous system regeneration.This review provides an overview of studies devoted to testing the effect of thyroid hormones on peripheral nerve regeneration.Also it emphasizes the importance of combining biodegradable tubes with local administration of triiodothyronine for future clinical therapy of human severe injured nerves.We highlight that the local and single administration of triiodothyronine within biodegradable nerve guide improves significantly the regeneration of severed peripheral nerves,and accelerates functional recovering.This technique provides a serious step towards future clinical application of triiodothyronine in human severe injured nerves.The possible regulatory mechanism by which triiodothyronine stimulates peripheral nerve regeneration is a rapid action on both axotomized neurons and Schwann cells.

Micro RNA-124 slows down the progression of Huntington's disease by promoting neurogenesis in the striatum

MicroRNA-124 contributes to neurogenesis through regulating its targets, but its expression both in the brain of Huntington＇s disease mouse models and patients is decreased. However, the effects of microRNA-124 on the progression of Huntington＇s disease have not been reported. Results from this study showed that microRNA-124 increased the latency to fall for each R6/2 Hunting- ton＇s disease transgenic mouse in the rotarod test. 5-Bromo-2＇-deoxyuridine （BrdU） staining of the striatum shows an increase in neurogenesis. In addition, brain-derived neurotrophic factor and peroxisome proliferator-activated receptor gamma coactivator 1-alpha protein levels in the striatum were increased and SRY-related HMG box transcription factor 9 protein level was de- creased. These findings suggest that microRNA-124 slows down the progression of Huntington＇s disease possibly through its important role in neuronal differentiation and survival.

Inhibition of LncRNA Vof-16 expression promotes nerve regeneration and functional recovery after spinal cord injury

Our previous RNA sequencing study showed that the long non-coding RNA ischemia-related factor Vof-16(lncRNA Vof-16)was upregulated after spinal cord injury,but its precise role in spinal cord injury remains unclear.Bioinformatics predictions have indicated that lncRNA Vof-16 may participate in the pathophysiological processes of inflammation and apoptosis.PC12 cells were transfected with a pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector to express an lncRNA Vof-16 knockdown lentivirus and a pHLV-CMVIE-ZsGree-Puro vector to express an lncRNA Vof-16 overexpression lentivirus.The overexpression of lncRNA Vof-16 inhibited PC12 cell survival,proliferation,migration,and neurite extension,whereas lncRNA Vof-16 knockdown lentiviral vector resulted in the opposite effects in PC12 cells.Western blot assay results showed that the overexpression of lncRNA Vof-16 increased the protein expression levels of interleukin 6,tumor necrosis factor-α,and Caspase-3 and decreased Bcl-2 expression levels in PC12 cells.Furthermore,we established rat models of spinal cord injury using the complete transection at T10.Spinal cord injury model rats were injected with the lncRNA Vof-16 knockdown or overexpression lentiviral vectors immediately after injury.At 7 days after spinal cord injury,rats treated with lncRNA Vof-16 knockdown displayed increased neuronal survival and enhanced axonal extension.At 8 weeks after spinal cord injury,rats treated with the lncRNA Vof-16 knockdown lentiviral vector displayed improved neurological function in the hind limb.Notably,lncRNA Vof-16 knockdown injection increased Bcl-2 expression and decreased tumor necrosis factor-αand Caspase-3 expression in treated animals.Rats treated with the lncRNA Vof-16 overexpression lentiviral vector displayed opposite trends.These findings suggested that lncRNA Vof-16 is associated with the regulation of inflammation and apoptosis.The inhibition of lncRNA Vof-16 may be useful for promoting nerve regeneration and functional recovery after spinal cord injury.The experiments were approved by the Institutional Animal Care and Use Committee of Guangdong Medical University,China.

A SteMNess perspective of survival motor neuron function： splicing factors in stem cell biology and disease

Genome-wide analyses of metazoan messenger RNA （mRNA） species are unveiling the extensive transcriptional diversity generated by alternative splicing （AS）. Research is also beginning to identify the splicing factors and AS events required to maintain the balance between stem cell renewal （i.e stemness properties） and differentiation. One set of proteins at the center of spliceosome biogenesis are the survival motor neuron （SMN） complex constituents, which have a critical role in the assembly of spliceosomal small nuclear ribonucleoproteins （snRNPs） in all cells. In this review we discuss what is currently known about how AS controls pluripotency and cell fate and consider how an increased requirement for splicing factors, including SMN, helps to maintain an enrichment of stem cell-specific AS events. Furthermore, we highlight studies showing that mutations in specific splicing factors can lead to the aberrant development, and cause targeted degeneration of the nervous system. Using SMN as an example, we discuss the perspective of how stem cell-specific changes in splicing factors can lead to developmental defects and the selective degeneration of particular tissues. Finally we consider the expanding role of SMN, and other splicing factors, in the regulation of gene expression in stem cell biology, thereby providing insight into a number of debilitating diseases.

Nusinersen,an exon 7 inclusion drug for spinal muscular atrophy:A minireview

Spinal muscular atrophy is an autosomal recessive neuromuscular disease with incidence of 1 in 5000 to 10000 live births and is produced by homozygous deletion of exons 7 and 8 in the SMN1 gene.The SMN1 and SMN2 genes encode the survival motor neuron protein,a crucial protein for the preservation of motor neurons.Use of the newer drug,Nusinersen,from early infancy has shown improvement in clinical outcomes of spinal muscular atrophy patients.

Mesenchymal stromal cell biotherapy for Parkinson’s disease premotor symptoms

Parkinson’s disease(PD)is a neurodegenerative disorder with motor deficits due to nigrostriatal dopamine depletion and with the non-motor/premotor symptoms(NMS)such as anxiety,cognitive dysfunction,depression,hyposmia,and sleep disorders.NMS is presented in at least one-fifth of the patients with PD.With the histological information being investigated,stem cells are shown to provide neurotrophic supports and cellular replacement in the damaging brain areas under PD conditions.Pathological change of progressive PD includes degeneration and loss of dopaminergic neurons in the substantia nigra of the midbrain.The current stem cell beneficial effect addresses dopamine boost for the striatal neurons and gliovascular mechanisms as competing for validated PD drug targets.In addition,there are clinical interventions for improving the patient’s NMS and targeting their autonomic dysfunction,dementia,mood disorders,or sleep problems.In our and many others’research using brain injury models,multipotent mesenchymal stromal cells demonstrate an additional and unique ability to alleviate depressive-like behaviors,independent of an accelerated motor recovery.Intranasal delivery of the stem cells is discussed for it is extensively tested in rodent animal models of neurological and psychiatric disorders.In this review,we attempt to discuss the repairing potentials of transplanted cells into parkinsonism pathological regions of motor deficits and focus on preventive and treatment effects.From new approaches in the PD biological therapy,it is believed that it can as well benefit patients against PD-NMS.

Compound heterozygous mutation in two unrelated cases of Chinese spinal muscular atrophy patients

Background Infantile proximal spinal muscular atrophy （SMA） is a common autosomal recessive neuromuscular disorder. Approximately 90-95% cases of SMA result from homozygous deletion of survival motor neuron gene 1（SMN1） and 5% cases are caused by compound heterozygous mutation （a SMN1 deletion on one allele and a subtle mutation on the other allele）.Methods In this research, two unrelated patients were clinically diagnosed according to the criteria of proximal SMA. Genetic diagnosis was performed to detect the homozygous deletion of exon 7 of SMN1 by PCR-restriction fragment length polymorphism （RFLP） and genomic sequencing. Multiplex ligation-dependent probe amplification （MLPA） analysis was carried out to measure copy numbers of SMN1, SMN2 and neuronal apoptosis inhibitor protein （NAIP） in the patients. Further sequencing of SMN1allele-specific PCR （AS-PCR） and SMN1 clones were also performed to analyze the point mutation of SMN1 gene. Additionally,the pedigree analysis of these two families was carried out to identify the transmission of the mutation.Results The inconsistent results using PCR-RFLP and genomic sequencing showed homozygous deletion of exon 7 of SMN1 and heterozygous deletion accompanied with a suspicious mutation in SMN1 gene, respectively. MLPA analysis of these two cases exhibited one SMN1 copy deletion. One identical c.863G〉T （p. Arg288Met） mutation was found in two cases by sequencing the SMN1 clones, which confirmed that both cases were SMA compound heterozygotes. One case showed partial conversion to form hybrid SMN （SMN2 17/SMN1 E8） identified by clones sequencing and another case carrying 3 SMN2 implied complete conversion from SMN1 to SMN2.Conclusion p. Arg288Met is more a disease-causing mutation than a polymorphism variation, and children with this mutation may have more severe phenotypes.

Association between SMN2 methylation and disease severity in Chinese children with spinal muscular atrophy

The homozygous loss of the survival motor neuron 1 （SMN1） gene is the primary cause of spinal muscular atrophy （SMA）, a neuromuscular degenerative disease. A genetically similar gene, SMN2, which is not functionally equivalent in all SMA patients, modifies the clinical SMA phenotypes. We analyzed the methylation levels of 4 CpG islands （CGIs） in SMN2 in 35 Chinese children with SMA by MassARRAY. We found that three CpG units located in CGI 1 （nucleotides （nt） -871, -735） and CGI 4 （nt ＋999） are significantly hypomethylated in SMA type III compared with type I or II children after receiving Bonferroni correction. In addition to the differentially methylated CpG unit of nt -871, the methylation level of the nt -290/-288/-285 unit was negatively correlated with the expression of SMN2 full-length transcripts （SMN2-fl）. In addition, the methylation level at nt ＋938 was inversely proportional to the ratio of SMN2-fl and lacking exon 7 transcripts （SMN2-A7, fl/A7）, and was not associated with the SMN2 transcript levels. Thus, we can conclude that SMN2 methylation may regulate the SMA disease phenotype by modulating its transcription.

Prenatal diagnosis of spinal muscular atrophy in Chinese by genetic analysis of fetal cells

Background Spinal muscular atrophy （SMA） is an autosomal recessive disease characterized by degeneration of anterior horn cells of the spinal cord. The survival motor neuron gene is SMA-determining gene deleted in approximately 95% of SMA patients. This study was undertaken to predict prenatal SMA efficiently and rapidly in families with previously affected child. Methods Prenatal diagnosis was made in 8 fetuses with a family history of SMA. Polymerase （PCR） and restriction fragment length polymorphism （RFLP） were used for the detection of the neuron gene. Results The survival motor neuron fetuses were detected positive and the gene was not found in 6 fetuses, ruling out the diagnosis of SMA. Two fetuses were detected positive and the pregnancies were terminated. Conclusion Our method is effective and convenient in prenatal diagnosis of SMA.

Deletion analysis of SMN1 and NAIP genes in southern Chinese children with spinal muscular atrophy

Spinal muscular atrophy （SMA） is a disorder characterized by degeneration of lower motor neurons and occasionally bulbar motor neurons leading to progressive limb and trunk paralysis as well as muscular atrophy. Three types of SMA are recognized depending on the age of onset, the maximum muscular activity achieved, and survivorship： SMA1, SMA2, and SMA3. The survival of motor neuron （SMN） gene has been identified as an SMA determining gene, whereas the neuronal apoptosis inhibitory protein （NAlP） gene is considered to be a modifying factor of the severity of SMA. The main objective of this study was to analyze the deletion of SMN1 and NAIP genes in southern Chinese children with SMA. Here, polymerase chain reaction （PCR） combined with restriction fragment length polymorphism （RFLP） was performed to detect the deletion of both exon 7 and exon 8 of SMN1 and exon 5 of NAIP in 62 southern Chinese children with strongly suspected clinical symptoms of SMA. All the 32 SMA1 patients and 76% （13/17） of SMA2 patients showed homozygous deletions for exon 7 and exon 8, and all the 13 SMA3 patients showed single deletion of SMNI exon 7 along with 24% （4/17） of SMA2 patients. Eleven out of 32 （34%） SMA1 patients showed NAIP deletion, and none of SMA2 and SMA3 patients was found to have NA1P deletion. The findings of homozygous deletions ofexon 7 and/or exon 8 ofSMN1 gene confirmed the diagnosis of SMA, and suggested that the deletion ofSMN1 exon 7 is a major cause of SMA in southern Chinese children, and that the NAIP gene may be a modifying factor for disease severity of SMAI. The molecular diagnosis system based on PCR-RFLP analysis can conveniently be applied in the clinical testing, genetic counseling, prenatal diagnosis and preimplantation genetic diagnosis of SMA.