Neuronal Neurite Extension Research Articles

A Novel Constitutively Active c . 98 G > C , p.(R33P) Variant in RAB11A Associated with Intellectual Disability Promotes Neuritogenesis and Affects Oligodendroglial Arborization

Whole exome sequencing/whole genome sequencing has accelerated the identification of novel genes associated with intellectual disabilities (ID), and RAB11A which encodes an endosomal small GTPase is among them. However, consequent neural abnormalities have not been studied, and pathophysiological mechanisms underlying the ID and other clinical features in patients harboring RAB11A variants remain to be clarified. In this study, we report a novel de novo missense variant in RAB11A, NM_004663.5: c . 98 G > C , which would result in NP_004654.1: p.(R33P) substitution, in a Japanese boy with severe ID and hypomyelination. Biochemical analyses indicated that the RAB11A-R33P is a gain-of-function, constitutively active variant. Accordingly, the introduction of the RAB11A-R33P promoted neurite extension in neurons like a known constitutively active variant Rab11A-Q70L. In addition, the RAB11A-R33P induced excessive branching with thinner processes in oligodendrocytes. These results indicate that the gain-of-function RAB11A-R33P variant in association with ID and hypomyelination affects neural cells and can be deleterious to them, especially to oligodendrocytes, and strongly suggest the pathogenic role of the RAB11A-R33P variant in neurodevelopmental impairments, especially in the hypomyelination.

Regulation of nerve cells using conductive nanofibrous scaffolds for controlled release of Lycium barbarum polysaccharides and nerve growth factor.

Currently, more and more patients suffer from peripheral nerve injury due to trauma, tumor and other causes worldwide. Biomaterial-based nerve conduits are increasingly recognized as a potential alternative to nerve autografts for the treatment of peripheral nerve injury. However, an ideal nerve conduit must offer topological guidance and biochemical and electrical signal transduction mechanisms. In this work, aligned conductive nanofibrous scaffolds comprising polylactic-co-glycolic acid and multiwalled carbon nanotubes (MWCNTs) were fabricated via coaxial electrospinning, and nerve growth factor (NGF) and Lycium barbarum polysaccharides (LBP) purified from the wolfberry were loaded on the core and shell layers of the nanofibers, respectively. LBP were confirmed to accelerate long-distance axon regeneration after severe peripheral nerve injury. In addition, the synergistic promotion of LBP and NGF on nerve cell proliferation and neurite outgrowth was demonstrated. MWCNTs were introduced into the aligned fibers to further increase the electrical conductivity, which promoted the directional growth and neurite extension of neurons in vitro. Further, the combination of conductive fibrous scaffolds with electrical stimulation that mimics endogenous electric fields significantly promoted the differentiation of PC12 cells and the axon outgrowth of neurons. Based on robust cell-induced behaviors, conductive composite fibers with optimized fiber alignment may be used for the promotion of nerve recovery.

Insulin enhances neurite extension and myelination of diabetic neuropathy neurons.

BackgroundThe authors established an in vitro model of diabetic neuropathy based on the culture system of primary neurons and Schwann cells (SCs) to mimic similar symptoms observed in in vivo models of this complication, such as impaired neurite extension and impaired myelination. The model was then utilized to investigate the effects of insulin on enhancing neurite extension and myelination of diabetic neurons.MethodsSCs and primary neurons were cultured under conditions mimicking hyperglycemia prepared by adding glucose to the basal culture medium. In a single culture, the proliferation and maturation of SCs and the neurite extension of neurons were evaluated. In a co-culture, the percentage of myelination of diabetic neurons was investigated. Insulin at different concentrations was supplemented to culture media to examine its effects on neurite extension and myelination.ResultsThe cells showed similar symptoms observed in in vivo models of this complication. In a single culture, hyperglycemia attenuated the proliferation and maturation of SCs, induced apoptosis, and impaired neurite extension of both sensory and motor neurons. In a co-culture of SCs and neurons, the percentage of myelinated neurites in the hyperglycemia-treated group was significantly lower than that in the control group. This impaired neurite extension and myelination was reversed by the introduction of insulin to the hyperglycemic culture media.ConclusionsInsulin may be a potential candidate for improving diabetic neuropathy. Insulin can function as a neurotrophic factor to support both neurons and SCs. Further research is needed to discover the potential of insulin in improving diabetic neuropathy.

PKA modulation of Rac in neuronal cells.

PKA modulation of Rac in neuronal cells.

Involvement of GPR12 in the induction of neurite outgrowth in PC12 cells

GPR12, an orphan G protein-coupled receptor, constitutively activates the Gs signaling pathway and further increases intracellular cyclic AMP. GPR12 overexpression has been reported to promote neurite extension in neurons or transform neuro2a neuroblastoma cells into neuron-like cells. However, the possible effects and mechanisms of GPR12 in the differentiation of PC12 cells are still unknown. The present study shows that GPR12 overexpression induced PC12 cells differentiation into neuron-like cells with enlarged cell sizes and neuritogenesis possibly via activation of Erk1/2 signaling and significantly increased the expression of several neurite outgrowth-related genes, including Bcl-xL, Bcl-2 and synaptophysin. These findings indicate that GPR12 may play a role in neurite outgrowth during PC12 cell differentiation.

Downregulation of thymosin β4 in neural progenitor grafts promotes spinal cord regeneration

Thymosin beta4 (Tbeta4) is an actin-binding peptide whose expression in developing brain correlates with migration and neurite extension of neurons. Here, we studied the effects of the downregulation of Tbeta4 expression on growth and differentiation of murine neural progenitor cells (NPCs), using an antisense lentiviral vector. In differentiation-promoting medium, we found twice the number of neurons derived from the Tbeta4-antisense-transduced NPCs, which showed enhanced neurite outgrowth accompanied by increased expression of the adhesion complex N-cadherin-beta-catenin and increased ERK activation. Importantly, when the Tbeta4-antisense-transduced NPCs were transplanted in vivo into a mouse model of spinal cord injury, they promoted a significantly greater functional recovery. Locomotory recovery correlated with increased expression of the regeneration-promoting cell adhesion molecule L1 by the grafted Tbeta4-antisense-transduced NPCs. This resulted in an increased number of regenerating axons and in sprouting of serotonergic fibers surrounding and contacting the Tbeta4-antisense-transduced NPCs grafted into the lesion site. In conclusion, our data identify a new role for Tbeta4 in neuronal differentiation of NPCs by regulating fate determination and process outgrowth. Moreover, NPCs with reduced Tbeta4 levels generate an L1-enriched environment in the lesioned spinal cord that favors growth and sprouting of spared host axons and enhances the endogenous tissue-repair processes.

Essential Role of Neural Wiskott-Aldrich Syndrome Protein in Neurite Extension in PC12 Cells and Rat Hippocampal Primary Culture Cells

Neural Wiskott-Aldrich syndrome protein (N-WASP) is an actin-regulating protein that induces filopodium formation downstream of Cdc42. It has been shown that filopodia actively extend from the growth cone, a guidance apparatus located at the tip of neurites, suggesting their role in neurite extension. Here we examined the possible involvement of N-WASP in the neurite extension process. Since verprolin, cofilin homology and acidic region (VCA) of N-WASP is known to be required for the activation of Arp2/3 complex that induces actin polymerization, we prepared a mutant (Deltacof) lacking four amino acid residues in the cofilin homology region. The corresponding residues in WASP had been reported to be mutated in some Wiskott-Aldrich syndrome patients. Expression of Deltacof N-WASP suppressed neurite extension of PC12 cells. In support of this, the VCA region of Deltacof cannot activate Arp2/3 complex enough compared with wild-type VCA. Furthermore, H208D mutant, which has been shown unable to bind to Cdc42, also works as a dominant negative mutant in neurite extension assay. Interestingly, the expression of H208D-Deltacof double mutant has no significant dominant negative effect. Finally, the expression of the Deltacof mutant also severely inhibited the neurite extension of primary neurons from rat hippocampus. Thus, N-WASP is thought to be a general regulator of the actin cytoskeleton indispensable for neurite extension, which is probably caused through Cdc42 signaling and Arp2/3 complex-induced actin polymerization.

Gene structure of mouse BIT/SHPS-1

BIT/SHPS-1/SIRPα/P84 is a unique molecule with a high degree of homology with immune antigen recognition molecules (immunoglobulin, T-cell receptor and MHC), and is highly expressed in the brain. The extracellular region contains three immunoglobulin-like domains (V-type, C1-type and C1-type), and the intracellular region contains two signalling motifs that interact with SHP-2 protein tyrosine phosphatase. BIT-coated plates support cell-substrate adhesion and neurite extension of neurons, and BIT participates in neuronal signal transduction. Diversity of the V-type domain sequences of human BIT has been reported. In the present study we analysed the structure of the mouse BIT gene (Bit). The protein coding region consists of eight exons corresponding to a signal peptide, a V-type domain, a C1-type domain, a C1-type domain, a transmembrane region and three parts of one cytoplasmic region. The two signalling motifs are encoded in one exon. Four splicing forms of mouse BIT were revealed. We also found the sequence diversity in three mouse strains, namely BALB/c, 129/Sv and C57BL/6. The substitution patterns of amino acids and nucleotides indicate positive pressure to alter the amino acids in the V-type domain in evolution. Immunoblot analyses showed that mouse BIT and human BITα are predominantly expressed in the brain. On the bases of these findings we discuss the possibility that BIT contributes to the genetic individuality and diversity of the brain.

Gene structure of mouse BIT/SHPS-1

BIT/SHPS-1/SIRPalpha/P84 is a unique molecule with a high degree of homology with immune antigen recognition molecules (immunoglobulin, T-cell receptor and MHC), and is highly expressed in the brain. The extracellular region contains three immunoglobulin-like domains (V-type, C1-type and C1-type), and the intracellular region contains two signalling motifs that interact with SHP-2 protein tyrosine phosphatase. BIT-coated plates support cell-substrate adhesion and neurite extension of neurons, and BIT participates in neuronal signal transduction. Diversity of the V-type domain sequences of human BIT has been reported. In the present study we analysed the structure of the mouse BIT gene (Bit). The protein coding region consists of eight exons corresponding to a signal peptide, a V-type domain, a C1-type domain, a C1-type domain, a transmembrane region and three parts of one cytoplasmic region. The two signalling motifs are encoded in one exon. Four splicing forms of mouse BIT were revealed. We also found the sequence diversity in three mouse strains, namely BALB/c, 129/Sv and C57BL/6. The substitution patterns of amino acids and nucleotides indicate positive pressure to alter the amino acids in the V-type domain in evolution. Immunoblot analyses showed that mouse BIT and human BITalpha are predominantly expressed in the brain. On the bases of these findings we discuss the possibility that BIT contributes to the genetic individuality and diversity of the brain.

Differential neurite growth on astrocyte substrates: interspecies facilitation in green fluorescent protein-transfected rat and human neurons

In the present study, we used co-culture of astrocytes from one species with neurons from a different species to examine neuritic outgrowth. We include a focus on human cells. Three types of neuron were used, including rat hippocampal dentate granule cells, rat hypothalamic neurons and human cortical neurons. To visualize neuronal processes, neurons were either immunostained with GABA antiserum or transfected with the jellyfish green fluorescent protein gene. The entire axonal and dendritic fields of single neurons could be quantitatively analysed based on their strong green fluorescent protein label. Astrocytes were obtained from rat hippocampus or hypothalamus, chicken cortex, normal human cortex, human cortex lesion, and from the sclerotic human hippocampus after surgery for intractable temporal lobe epilepsy. In the absence of astrocytes, isolated neurons died within three to four days. In contrast, neurons from both rat and human brains survived and extended dendrites and axons on rat, chicken and human astrocytes or in their conditioned medium. Astrocytes from interspecies cultures were not only capable of enhancing the survival of neuron co-cultures, but neuronal neurite extension in some cases was even greater on heterospecific astrocytes than on homospecific astrocytes. To support the hypothesis that synaptogenesis of rat hippocampal neurons was accelerated by a substrate of human astrocytes, we used a functional assay based on time-lapse confocal laser or digital imaging of calcium responses to transmitter release; synaptic responses were found earlier when rat neurons were grown on rat or human astrocytes than in the absence of these astrocytes. These data indicate that rodent glial cells enhance human neurite extension, and that rat neurite outgrowth can be used as a type of bioassay for the neurite promoting capacity of different derivations of human glia.

An in vitro model to study diabetic neuropathy

Adult dorsal root ganglion (DRG) neurons from diabetic and normal C57BL mice were cultured and compared for survival and neurite extension. Neurons from diabetic mice have improved their survival and neurite extension when the insulin concentration was increased but it was never as good as that of controls. The increase of glucose concentration in serum-free media to ten times higher than normal improved both survival and neurite extension of neurons from diabetic animals.