Unveiling Neuroprotection and Regeneration Mechanisms in Optic Nerve Injury: Insight from Neural Progenitor Cell Therapy with Focus on Vps35 and Syntaxin12.

Axonal degeneration resulting from optic nerve damage can lead to the progressive death of retinal ganglion cells (RGCs), culminating in irreversible vision loss. We contrasted two methods for inducing optic nerve damage: optic nerve compression (ONCo) and optic nerve crush (ONCr). These were assessed for their respective merits in simulating traumatic optic neuropathies and neurodegeneration. We also administered neural progenitor cells (NPCs) into the subtenon space to validate their potential in mitigating optic nerve damage. Our findings indicate that both ONCo and ONCr successfully induced optic nerve damage, as shown by increases in ischemia and expression of genes linked to neuronal regeneration. Post NPC injection, recovery in the expression of neuronal regeneration-related genes was more pronounced in the ONCo model than in the ONCr model, while inflammation-related gene expression saw a better recovery in ONCr. In addition, the proteomic analysis of R28 cells in hypoxic conditions identified Vps35 and Syntaxin12 genes. Vps35 preserved the mitochondrial function in ONCo, while Syntaxin12 appeared to restrain inflammation via the Wnt/ß-catenin signaling pathway in ONCr. NPCs managed to restore damaged RGCs by elevating neuroprotection factors and controlling inflammation through mitochondrial homeostasis and Wnt/ß-catenin signaling in hypoxia-injured R28 cells and in both animal models. Our results suggest that ischemic injury and crush injury cause optic nerve damage via different mechanisms, which can be effectively simulated using ONCo and ONCr, respectively. Moreover, cell-based therapies such as NPCs may offer promising avenues for treating various optic neuropathies, including ischemic and crush injuries.

Delayed hair cycle in mnd2 mutant mice lacking HtrA2 serine protease activity.

The premature death and degeneration of striatal neurons are typical hallmarks of HtrA2-inactivated motor neuron degeneration 2 (mnd2) mice. Although HtrA2 has been extensively studied in relation to the regulation of apoptosis using mnd2 mice, little is known about the other physiological functions of HtrA2. In this study, we found that the skin color of wild-type (WT) and mnd2 mice was black and pink on postnatal day 32. Using histological and molecular assays (i.e., assessing the activation of MAPK and expression patterns of PCNA), we demonstrated that this differential skin color change is consistent with the delay in the telogen - to - anagen phase of the hair cycle in mnd2 mice. We also examined adipocytes in the subcutaneous skin layer, finding that HtrA2 inactivation leads to the growth retardation of adipocytes, thereby delaying the hair cycle of mnd2 mice. Collectively, these findings show for the first time that HtrA2 plays an essential role in regulating the adipogenesis-associated hair cycle.

The Role of Extracellular Vesicles in Optic Nerve Injury: Neuroprotection and Mitochondrial Homeostasis.

Stem cell therapies hold great promise as alternative treatments for incurable optic nerve disorders. Although mesenchymal stem cells exhibit various tissue regeneration and recovery capabilities that may serve as valuable therapies, the clinical applications remain limited. Thus, we investigated the utility of extracellular vesicles (EVs) from human placenta-derived mesenchymal stem cells (hPSCs) in this context. Hypoxically preconditioned hPSCs (HPPSCs) were prepared via short-term incubation under 2.2% O2 and 5.5% CO2. The EVs were then isolated. R28 cells (retinal precursor cells) were exposed to CoCl2 and treated with EVs for 24 h. Cell proliferation and regeneration were measured using a BrdU assay and immunoblotting; ATP quantification revealed the extent of the mitochondrial function. The proteome was determined via liquid chromatography-tandem mass spectroscopy. Differentially expressed proteins (DEPs) were detected and their interactions identified. HPPSC_EVs functions were explored using animal models of optic nerve compression. HPPSC_EVs restored cell proliferation and mitochondrial quality control in R28 cells damaged by CoCl2. We identified DEPs (p < 0.05) that aided recovery. The mitochondrial DEPs included LONP1; PARK7; VDAC1, 2, and 3; HSPD1; and HSPA9. EVs regulated the levels of mitophagic proteins in R28 cells injured by hypoxia; the protein levels did not increase in LONP1 knockdown cells. LONP1 is a key mediator of the mitophagy that restores mitochondrial function after hypoxia-induced optic nerve injury.

TGFß-Treated Placenta-Derived Mesenchymal Stem Cells Selectively Promote Anti-Adipogenesis in Thyroid-Associated Ophthalmopathy.

Orbital fibroblasts (OFs) in thyroid-associated ophthalmopathy (TAO) are differentiated from pre-adipocytes and mature adipocytes; increased lipid and fat expansion are the major characteristics of ophthalmic manifestations. Human placental mesenchymal stem cells (hPMSCs) were reported to immunomodulate pathogenesis and suppress adipogenesis in TAO OFs. Here, we prepared transforming growth factor ß (TGFß, 20 ng/mL)-treated hPMSCs (TGFß-hPMSCs) in order to enhance anti-adipogenic effects in vitro and in TAO mice. TAO OFs were grown in a differentiation medium and then co-cultured with hPMSCs or TGFß-hPMSCs. TAO OFs were analyzed via quantitative real-time polymerase chain reaction, Oil red O staining, and western blotting. The results showed that TGFß-hPMSCs reduced the expression of adipogenic, lipogenic, and fibrotic genes better than hPMSCs in TAO OFs. Moreover, the adipose area decreased more in TAO mice injected with TGFß-hPMSCs compared to those injected with hPMSCs or a steroid. Further, TGFß-hPMSCs inhibited inflammation as effectively as a steroid. In conclusion, TGFß-hPMSCs suppressed adipogenesis and lipogenesis in vitro and in TAO mice, and the effects were mediated by the SMAD 2/3 pathways. Furthermore, TGFß-hPMSCs exhibited anti-inflammatory and anti-fibrotic functions, which suggests that they could be a new and safe method to promote the anti-adipogenic function of hPMSCs to treat TAO patients.

Human Pluripotent Stem Cell-Derived Neural Progenitor Cells Promote Retinal Ganglion Cell Survival and Axon Recovery in an Optic Nerve Compression Animal Model.

Human pluripotent stem cell-derived neural progenitor cells (NPCs) have the potential to recover from nerve injury. We previously reported that human placenta-derived mesenchymal stem cells (PSCs) have neuroprotective effects. To evaluate the potential benefit of NPCs, we compared them to PSCs using R28 cells under hypoxic conditions and a rat model of optic nerve injury. NPCs and PSCs (2 × 106 cells) were injected into the subtenon space. After 1, 2, and 4 weeks, we examined changes in target proteins in the retina and optic nerve. NPCs significantly induced vascular endothelial growth factor (Vegf) compared to age-matched shams and PSC groups at 2 weeks; they also induced neurofilaments in the retina compared to the sham group at 4 weeks. In addition, the expression of brain-derived neurotrophic factor (Bdnf) was high in the retina in the NPC group at 2 weeks, while expression in the optic nerve was high in both the NPC and PSC groups. The low expression of ionized calcium-binding adapter molecule 1 (Iba1) in the retina had recovered at 2 weeks after NPC injection and at 4 weeks after PSC injection. The expression of the inflammatory protein NLR family, pyrin domain containing 3 (Nlrp3) was significantly reduced at 1 week, and that of tumor necrosis factor-α (Tnf-α) in the optic nerves of the NPC group was lower at 2 weeks. Regarding retinal ganglion cells, the expressions of Brn3a and Tuj1 in the retina were enhanced in the NPC group compared to sham controls at 4 weeks. NPC injections increased Gap43 expression from 2 weeks and reduced Iba1 expression in the optic nerves during the recovery period. In addition, R28 cells exposed to hypoxic conditions showed increased cell survival when cocultured with NPCs compared to PSCs. Both Wnt/ß-catenin signaling and increased Nf-ĸb could contribute to the rescue of damaged retinal ganglion cells via upregulation of neuroprotective factors, microglial engagement, and anti-inflammatory regulation by NPCs. This study suggests that NPCs could be useful for the cellular treatment of various optic neuropathies, together with cell therapy using mesenchymal stem cells.

ß2-adrenergic receptor expression and the effects of norepinephrine and propranolol on various head and neck cancer subtypes.

The present study aimed to investigate expression of ß2-adrenergic receptor (AR), the effect of the stress-related neurotransmitter norepinephrine (NE) on cell viability, proliferation and the therapeutic effect of propranolol, which is a typical ß-blocker in various type of head and neck cancers for the first time. The ß2-AR expression was investigated using immunohistochemistry and an immunoreactive scoring (IRS) system in 57 different head and neck cancer specimens, and reverse transcriptase-polymerase chain reaction and western blotting in four head and neck cancer cell lines (HNCCLs). Cell viability and proliferation assays were performed using 0, 1, 5 and 10 µM of NE and 1 µM of propranolol in four HNCCLs. The expression of ß2-AR was positive in the majority of head and neck cancer tissues (55/57, 96.5%); however, it was significantly higher in oral cavity cancer than in pharyngeal cancer (median IRS: 9 vs. 3; P<0.001). All HNCCLs exhibited ß2-AR expression, with a higher expression level detected in the oral cavity cancer cell line than in the others. NE stimulated viability (oral cavity, 206%; larynx, 156%; pharynx, 130%; nasal cavity, 137%; 10 µM NE) and proliferation (124, 176, 131 and 127%, respectively) in a dose-dependent manner in all HNCCLs. Conversely, propranolol attenuated such viability (55, 42, 18 and 22%, respectively) and proliferation (22, 40, 61 and 48%, respectively). In conclusion, the viability and proliferation of various head and neck cancers may be directly stimulated by stress and this may be attenuated by ß-blockers.

Essential roles of mitochondrial depolarization in neuron loss through microglial activation and attraction toward neurons.

As life spans increased, neurodegenerative disorders that affect aging populations have also increased. Progressive neuronal loss in specific brain regions is the most common cause of neurodegenerative disease; however, key determinants mediating neuron loss are not fully understood. Using a model of mitochondrial membrane potential (ΔΨm) loss, we found only 25% cell loss in SH-SY5Y (SH) neuronal mono-cultures, but interestingly, 85% neuronal loss occurred when neurons were co-cultured with BV2 microglia. SH neurons overexpressing uncoupling protein 2 exhibited an increase in neuron-microglia interactions, which represent an early step in microglial phagocytosis of neurons. This result indicates that ΔΨm loss in SH neurons is an important contributor to recruitment of BV2 microglia. Notably, we show that ΔΨm loss in BV2 microglia plays a crucial role in microglial activation and phagocytosis of damaged SH neurons. Thus, our study demonstrates that ΔΨm loss in both neurons and microglia is a critical determinant of neuron loss. These findings also offer new insights into neuroimmunological and bioenergetical aspects of neurodegenerative disease.

Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors.

The aim of this study was to produce dopaminergic neurons in vitro from human embryonic stem (hES) cells following treatment of various neurotrophic factors. MB03 hES cells were induced by retinoic acid (RA) or basic fibroblast growth factor (bFGF), which were further treated with brain derived neurotrophic factor (BDNF) or transforming growth factor (TGF)-alpha in each induction method during neuron differentiation days. At the final differentiation stage (21 days), all treatment groups revealed very similar levels (bFGF, 76-78%; RA, 70-74%) of mature neurons (anti-NF-200) in two induction methods irrespective of the addition of BDNF or TGF-alpha. In addition, immunostaining and HPLC analyses revealed higher levels of tyrosine hydroxylase (20+/-2.3%) and dopamine (265.5+/-62.8 pg/ml) in the bFGF- and TGF-alpha-treated hES cells than in RA- or BDNF-treated hES cells. These data are one of the first reports on the generation of dopaminergic neurons of hES cells in vitro. Also, our results indicate that TGF-alpha may be successfully used in the bFGF induction protocol and yield higher numbers of dopaminergic neurons from hES cells.