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PLXNA4 Gene
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This entry is adapted from the peer-reviewed paper 10.3390/ijms23031522

PLXNA4 gene, is known to play a role as a guide for axons in the development of the nervous system. When the PLXNA4 recombinant protein was added, neuron-related genes were increased. In the PLXNA4 gene knockdown experiment, the expression of neuron-related genes was not changed by LISSV exposure. The PLXNA4 gene is activated by sema family ligands. 

hUC-MSCs PLXNA4 Low-Intensity Sub-Sonic Vibration

1. Introduction

Plexin-A4 (encoded by PLXNA4) belongs to the plexin A family. It binds neuropilin to propagate signals with class 3 semaphorins (SEMA3) into neurons and plays an important role in the induction of axons to their synaptic target during neural development [1][2][3][4]. This complex activates the cdk-5-mediated isoform A of phosphatidylinositol 3-kinase enhancer and promotes glioma cell invasion through the modulation of Akt activity [5][6]. Plxna4 variants were found to be involved in Alzheimer’s disease pathogenesis through amyloid beta deposition [7].

2. Induction of PLXNA4 Gene during Neural Differentiation in Human Umbilical-Cord-Derived Mesenchymal Stem Cells by Low-Intensity Sub-Sonic Vibration

RNA sequencing is a very useful technique for elucidating the presence and sequences of RNA in a sample using next-generation sequencing. This technique shows changes in the cellular transcriptome at a given moment. After 4 days of exposure to LISSV, the hUC-MSCs changed their shape to a neuron-like morphology, at which time the cells were harvested for RNA sequencing. The PLXNA4 gene belongs to the plexin A family, and plexin A is a neuronal semaphorin receptor involved in axon guidance during neural development and neuron migration to synaptic organization [8][9][10][11]. In neurons, semaphorins transduce activation signals through plexin receptor proteins and the neuropilin family [12]. The recombinant PLXNA4 protein was used to study the role of the PLXNA4 gene in hUC-MSC differentiation by LISSV. The PLXNA4 gene was well expressed when 1.5 g or 2.0 g per well was added to hUC-MSCs. Cell proliferation was inhibited without dead cells. Thus, the reduction of cells by the PLXNA4 protein in the MTT assay indicates that hUC-MSCs have entered the differentiation process. After 5 days, a morphological change was induced and the morphology was close to that of a neuron. The cell body shone brightly, and two filaments extended in both directions around the cell body. Neuronal differentiation-related proteins such as MAP2, NEUROD1, and NF-L were expressed strongly in the immunofluorescence analysis, and the same results were confirmed in real-time PCR assays. These proteins and genes were also expressed in hUC-MSCs by LISSV [13], and the morphology of the differentiated cells was very similar in both cases. We observed that the differentiation process of hUC-MSCs induced by LISSV and PLXNA4 was slightly different. When LISSV was given to hUC-MSCs, differentiation of hUC-MSCs was a neural non-specific process, so all three types of neural cell markers, astrocytes, oligodendrocytes, and neurons, were induced. However, PLXNA4-induced differentiation in hUC-MSCs is a neuron-specific process, as GFAP and MBP were not expressed. Voltage-gated calcium channels are the main mediators that allow calcium to flow into neurons when depolarization occurs. The Cav2.1 is a P/Q-type calcium channel, and Cav2.2 is an N-type calcium channel, while P/Q and N channels trigger neurotransmitter release [14]. The recombinant PLXNA4 protein induced neural differentiation while expressing Cav2.1 and Cav2.2（Figures 1-7).
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Figure 1. Morphological changes after recombinant PLXNA4 protein treatment for 5 days in human umbilical-cord-derived mesenchymal stem cells. (a1,a2): Untreated control cells. (b1,b2): 1.5 μg of protein-treated cells. (c1,c2): 2.0 μg of protein-treated cells. Original magnification 40×.
Ijms 23 01522 g005Figure 2. Expression of neuron-related proteins and genes in human umbilical-cord-derived mesenchymal stem cells after recombinant PLXNA4 protein treatment. Cells were harvested 5 days after 2 μg/mL recombinant PLXNA4 protein treatment. (a) Fluorescence images of each protein. Original magnification 200×. (b) Fold expression of each gene using real-time polymerase chain reaction analysis. MAP2: Microtubule-associated protein 2. NEUROD1: Neuronal Differentiation 1, NF-L: Neurofilament-L, MBP: Myelin basic protein, Column heights correspond to mean values and error bars to standard deviations (n = 3). * p < 0.05, ** p < 0.01.
Ijms 23 01522 g006Figure 3. Expression of calcium channels in human umbilical-cord-derived mesenchymal stem cells after recombinant PLXNA4 protein treatment. Cells were harvested 5 days after 2 μg/mL recombinant PLXNA4 protein treatment. Fold expression of each gene using real-time polymerase chain reaction analysis. Cav2.1: Voltage-gated P/Q type calcium channel. Cav2.2: Voltage-gated N-type calcium channel, Column heights correspond to mean values and error bars to standard deviations (n = 3). ** p < 0.01.
hUC-MSCs are cells capable of self-renewal and differentiation into various lineages [15]. Wharton’s jelly derived from the human umbilical cord contains a higher amount of primitive MSCs compared to MSCs derived from bone marrow [16]. When MSCs begin to differentiate into cells of other lineages, those cells cannot maintain their stemness. Human MSCs express CD73, CD90, and CD105, but not CD34, CD45, or CD14 [17][18]. During both LISSV and recombinant PLXNA4 protein treatment, the expression of hUC-MSC specific markers, CD73 and CD105, was reduced. Therefore, those cells have begun to differentiate into other cells, particularly nerve-like cells.
To validate the function of the PLXNA4 gene, PLXNA4 gene silencing using siRNA analysis was performed. This assay was transient, and only 20–27 base pairs were used for gene silencing. After transfection of a specific gene base pair for an interfering RNA, the expression of the specific gene interferes with the complementary nucleotide sequence so the mRNA is degraded after transcription. In the case of LISSV exposure, PLXNA4 gene expression increased only in the silencing Negative (siNegative) expression group, and no change took place in the silencing PLXNA4 (siPLXNA4) expression group. The same results were observed in the morphological change data, and in the case of silencing PLXNA4 (siPLXNA4) expression, no morphological change occurred after exposure to LISSV. To confirm the neuronal differentiation after silencing PLXNA4 (siPLXNA4) expression in hUC-MSCs, we analyzed the neuron-related gene expression by LISSV. After silencing PLXNA4 (siPLXNA4) expression, the expression of neuron-related genes, NF-L, MBP, and MAP2, was unchanged compared to the controls and silencing Negative (siNegative) without LISSV groups. Consequently, we established that the PLXNA4 gene is associated with the neural differentiation of hUC-MSCs by LISSV.
Ijms 23 01522 g009Figure 4. Expression of neuron-associated genes in human umbilical-cord-derived mesenchymal stem cells after siPLXNA4 duplex transfection using real-time polymerase chain reaction analysis. Cells were harvested 4 days after low-intensity sub-sonic vibration treatment. NF-L: Neurofilament-L, MBP: Myelin basic protein, MAP2: Microtubule-associated protein 2. Column heights correspond to mean values and error bars to standard deviations (n = 3). * p < 0.05, ** p < 0.01.
The interaction of the neuropilin (Nrp)/plexin receptor complex and semaphorins plays an important role in axonal development in the central nervous system. This complex is also involved in a variety of other developmental processes, spanning from cell polarization to migration to neuronal maturation [19]. The semaphorin protein family consists of eight classes and is found in vertebrates and invertebrates. Plexin receptors for SEMA are classified into four classes, and plexin-A4 interacts with specific SEMA classes to mediate signal activation. The Nrp/PLXNA4 receptor complex interacts with class 3 and class 6 semaphorins and is involved in axon guidance and anti-angiogenesis when interacting with class 3 semaphorins [20][21]. SEMA3A, one of the class 3 semaphorins, interacts with plexins-A1 and -A4 to induce cytoskeletal disruption, the inhibition of cell proliferation, and adhesion in endothelial cells [22]. SEMA6A acts as a chemical repellent for sympathetic axons and is involved in lamina-specific axon formation in the hippocampus [23][24]. To identify SEMAs interacting with PLXNA4 in hUC-MSCs by LISSV, the researchers analyzed the gene expression of SEMA3A and SEMA6A after LISSV exposure. The researchers found that SEMA3A is upregulated by 5-fold, but the level of SEMA6A is not changed. When the Nrp/plxna4 receptor complex interacted with SEMA3A, its downstream molecule, FYN, is recruited for signal activation, which is involved in the process of collapse for cell differentiation [25]. The researchers confirmed that the FYN protein increases in a time-dependent manner. These results showed that increased levels of PLXNA4 expression induced by LISSV in hUC-MSCs are associated with an increase in SEMA3A, and that neuronal differentiation of hUC-MSCs induced by LISSV is thought to be due to semaphorin 3A–plexin-A4-dependent signaling activation.
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Figure 5. Expression of PLXNA4-dependent semaphorin signaling molecules in human umbilical-cord-derived mesenchymal stem cells by low-intensity sub-sonic vibration using real-time polymerase chain reaction analysis. Cells were harvested 4 days after low-intensity sub-sonic vibration treatment. SEMA3A: Semaphorin 3A, SEMA6A: Semaphorin 6A. Column heights correspond to mean values and error bars to standard deviations (n = 3).Ijms 23 01522 g013Figure 6. Expression of PLXNA4-dependent semaphorin signaling protein in human umbilical-cord-derived mesenchymal stem cells by low-intensity sub-sonic vibration. FYN: Src Family of nonreceptor tyrosine kinase p59.
Sema3A regulates the density of the dendritic spine, small membrane protrusions from dendrites of neurons [26][27], and this signaling activation induces growth cone collapse and neuronal cells [19][28]. SYN1 and Synaptophysin are presynaptic vesicle proteins located in the cytoplasmic membrane of the presynapse, and GAP43, a neuromodulin, plays a role in regulating presynaptic vesicular function and axonal growth [29][30]. The expression of SYN1, GAP43, and synaptophysin was induced in hUC-MSCs through sema3 signaling activation upon treatment with LISSV and the recombinant PLXNA4 protein.
Ijms 23 01522 g014Figure 7. Expression of presynaptic vesicle protein-associated genes in human umbilical-cord-derived mesenchymal stem cells after low-intensity sub-sonic vibration versus recombinant PLXNA4 protein. Cells were harvested 4 days after low-intensity sub-sonic vibration treatment and 5 days after recombinant PLXNA4 protein treatment. The fold change in the expression of each gene was analyzed using real-time polymerase chain reaction. SYN1: Synapsin 1, GAP43: Growth-Associated Protein 43, Column heights correspond to mean values and error bars to standard deviations (n = 3). * p < 0.05, ** p < 0.01.
This neuronal differentiation was activated through the Nrp/plxna4 receptor complex with the SEMA3A-dependent signaling mechanism. Discovering the specific mechanism that induces neural differentiation in hUC-MSCs via LISSV and applying it to neurodegenerative disorders will be very useful in stem cell therapy.

References

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Subjects: Biochemistry & Molecular Biology
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