SHED-CM for ALS Treatment

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder, characterized by the loss of upper and lower motor neurons, for which an effective treatment has yet to be developed. Previous reports have shown that excessive oxidative stress, related to mitochondrial dysfunction and the accumulation of misfolding protein, contributes to ALS pathology. In terms of treatment, it remains necessary to identify effective medicines for multiple therapeutic targets and have additive effects against several disorders. In this study, we investigated stem cells from human exfoliated deciduous teeth (SHED), which release many factors, such as neurotrophic factors and cytokines, and are applied to treat neurological diseases. Specifically, we examined whether SHED-conditioned medium (CM), i.e., the serum-free culture supernatant of SHED, reduced mutant SOD1-induced intracellular aggregates and neurotoxicity. We found that SHED-CM significantly suppressed the mutant SOD1-induced intracellular aggregates and neurotoxicity. The neuroprotective effects of SHED-CM are partly related to heat shock protein and the activation of insulin-like growth factor-1 receptor. SHED-CM also had a protective effect on induced pluripotent stem cell-derived motor neurons. Moreover, SHED-CM was effective against not only familial ALS but also sporadic ALS. Overall, these results suggest that SHED-CM could be a promising treatment for slowing the progression of ALS

Neuroprotective effect of 5-aminolevulinic acid against low inorganic phosphate in neuroblastoma SH-SY5Y cells

PiT-1 (encoded by SLC20A1) and PiT-2 (encoded by SLC20A2) are type-III sodium-dependent phosphate cotransporters (NaPiTs). Recently, SLC20A2 mutations have been found in patients with idiopathic basal ganglia calcification (IBGC), and were predicted to bring about an inability to transport Pi from the extracellular environment. Here we investigated the effect of low Pi loading on the human neuroblastoma SH-SY5Y and the human glioblastoma A172 cell lines. The results show a different sensitivity to low Pi loading and differential regulation of type-III NaPiTs in these cells. We also examined whether 5-aminolevulinic acid (5-ALA) inhibited low Pi loading-induced neurotoxicity in SH-SY5Y cells. Concomitant application of 5-ALA with low Pi loading markedly attenuated low Pi-induced cell death and mitochondrial dysfunction via the induction of HO-1 by p38 MAPK. The findings provide us with novel viewpoints to understand the pathophysiology of IBGC, and give a new insight into the clinical prevention and treatment of IBGC

A Golgi-targeting fluorescent probe for labile Fe(ii) to reveal an abnormal cellular iron distribution induced by dysfunction of VPS35

Iron is involved in numerous physiologically essential processes in our body. However, excessive iron is a pathogenic factor in neurodegenerative diseases, causing aberrant oxidative stress. Divalent metal transporter 1 (DMT1) acts as a primary transporter of Fe(II) ions. The intracellular delivery of DMT1 toward the cellular membrane via the trans-Golgi network during the endocytotic process is partially regulated by a retromer-mediated protein-sorting system comprising vacuolar protein-sorting proteins (VPSs). Thus, together with DMT1, the Golgi-apparatus acts as a hub organelle in the delivery system for intracellular Fe(II) ion. Dysfunction of the VPS-relevant protein sorting system can induce the abnormal delivery of DMT1 toward lysosomes concomitantly with Fe(II) ions. To explore this issue, we developed a fluorescent probe, Gol-SiRhoNox for the Golgi-specific detection of Fe(II) ions by integrating our original N-oxide-based Fe(II)-specific chemical switch, a new Golgi-localizable chemical motif, and polarity-sensitive fluorogenic scaffold. Our synchronous imaging study using Gol-SiRhoNox and LysoRhoNox, a previously developed fluorescent probe for lysosomal Fe(II), revealed that the intracellular distribution balance of Fe(II) ions between Golgi apparatus and lysosomes is normally Golgi-dominant, whereas the lysosome-specific elevation of Fe(II) ions was observed in cells with induced dysfunction of VPS35, a member of the retromer complex. Treatment of cells with dysfunctional VPS35 with R55, a molecular chaperone, resulted in the restoration of the subcellular distribution of Fe(II) ions to the Golgi-dominant state. These results indicate that the impairment of the DMT1 traffic machinery affects the subcellular iron homeostasis, promoting Fe(II) leakage at Golgi and lysosomal accumulation of Fe(II) through missorting of DMT1

Induced pluripotent stem cells derived from a patient with familial idiopathic basal ganglia calcification (IBGC) caused by a mutation in SLC20A2 gene

Idiopathic basal ganglia calcification (IBGC), also known as Fahr disease or primary familial brain calcifications (PFBC), is a rare neurodegenerative disorder characterized by calcium deposits in basal ganglia and other brain regions, causing neuropsychiatric and motor symptoms. We established human induced pluripotent stem cells (iPSCs) from an IBGC patient. The established IBGC-iPSCs carried SLC20A2 c.1848G > A mutation (p.W616* of translated protein PiT2), and also showed typical iPSC morphology, pluripotency markers, normal karyotype, and the ability of in vitro differentiation into three-germ layers. The iPSC line will be useful for further elucidating the pathomechanism and/or drug development for IBGC

The Novel gem-Dihydroperoxide 12AC3O Suppresses High Phosphate-Induced Calcification via Antioxidant Effects in p53LMAco1 Smooth Muscle Cells

The excessive intake of phosphate (Pi), or chronic kidney disease (CKD), can cause hyperphosphatemia and eventually lead to ectopic calcification, resulting in cerebrovascular diseases. It has been reported that reactive oxygen species (ROS), induced by high concentrations of Pi loading, play a key role in vascular calcification. Therefore, ROS suppression may be a useful treatment strategy for vascular calcification. 12AC3O is a newly synthesized gem-dihydroperoxide (DHP) that has potent antioxidant effects. In the present study, we investigated whether 12AC3O inhibited vascular calcification via its antioxidative capacity. To examine whether 12AC3O prevents vascular calcification under high Pi conditions, we performed Alizarin red and von Kossa staining, using the mouse aortic smooth muscle cell line p53LMAco1. Additionally, the effect of 12AC3O against oxidative stress, induced by high concentrations of Pi loading, was investigated using redox- sensitive dyes. Further, the direct trapping effect of 12AC3O on reactive oxygen species (ROS) was investigated by ESR analysis. Although high concentrations of Pi loading exacerbated vascular smooth muscle calcification, calcium deposition was suppressed by the treatment of both antioxidants and 12AC3O, suggesting that the suppression of ROS may be a candidate therapeutic approach for treating vascular calcification induced by high concentrations of Pi loading. Importantly, 12AC3O also attenuated oxidative stress. Furthermore, 12AC3O directly trapped superoxide anion and hydroxyl radical. These results suggest that ROS are closely involved in high concentrations of Pi-induced vascular calcification and that 12AC3O inhibits vascular calcification by directly trapping ROS

Ezrin Mediates Neuritogenesis via Down-Regulation of RhoA Activity in Cultured Cortical Neurons

<div><p>Neuronal morphogenesis is implicated in neuronal function and development with rearrangement of cytoskeletal organization. Ezrin, a member of Ezrin/Radixin/Moesin (ERM) proteins links between membrane proteins and actin cytoskeleton, and contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. Down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (<i>Vil2^kd/kd</i>) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (<i>Vil2^+/+</i>) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the <i>Vil2^kd/kd</i> mice embryo exhibited impairment of neuritogenesis. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the <i>Vil2^kd/kd</i> neurons. In addition, inhibition of Rho kinase and myosin II rescued the impairment of neuritogenesis in the <i>Vil2^kd/kd</i> neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons through down-regulation of RhoA activity.</p></div