Functional evaluation of PDGFB-variants in idiopathic basal ganglia calcification, using patient-derived iPS cells.

Causative genes in patients with idiopathic basal ganglia calcification (IBGC) (also called primary familial brain calcification (PFBC)) have been reported in the past several years. In this study, we surveyed the clinical and neuroimaging data of 70 sporadic patients and 16 families (86 unrelated probands in total) in Japan, and studied variants of PDGFB gene in the patients. Variant analyses of PDGFB showed four novel pathogenic variants, namely, two splice site variants (c.160 + 2T > A and c.457-1G > T), one deletion variant (c.33_34delCT), and one insertion variant (c.342_343insG). Moreover, we developed iPS cells (iPSCs) from three patients with PDGFB variants (c.160 + 2T > A, c.457-1G > T, and c.33_34 delCT) and induced endothelial cells. Enzyme-linked immunoassay analysis showed that the levels of PDGF-BB, a homodimer of PDGF-B, in the blood sera of patients with PDGFB variants were significantly decreased to 34.0% of that of the control levels. Those in the culture media of the endothelial cells derived from iPSCs of patients also significantly decreased to 58.6% of the control levels. As the endothelial cells developed from iPSCs of the patients showed a phenotype of the disease, further studies using IBGC-specific iPSCs will give us more information on the pathophysiology and the therapy of IBGC in the future.

SLC20A2 variants cause dysfunctional phosphate transport activity in endothelial cells induced from Idiopathic Basal Ganglia Calcification patients-derived iPSCs.

Idiopathic Basal Ganglia Calcification (IBGC) is a rare neuropsychiatric illness also known as Fahr's disease or Primary Familial Brain Calcification (PFBC). IBGC is caused by SLC20A2 variants, which encodes the inorganic phosphate (Pi) transporter PiT-2, a transmembrane protein associated with Pi homeostasis. We have reported novel SLC20A2 variants in the Japanese population and established an induced pluripotent stem cells (iPSCs) from an IBGC patient carrying a SLC20A2 variant. To investigate the effect of these SLC20A2 variants identified in our previous study, we used Chinese hamster ovary (CHO) cells expressing these variant proteins using the Flp-In system (Flp-In CHO cells), and showed that variant SLC20A2 proteins significantly disrupted the Pi transport activity in Flp-In CHO cells. Endothelial cells (ECs) represent important target cells for elucidating the pathology of IBGC. Using patient-derived iPSCs in this study, we differentiated these cells into ECs and found no significant difference in their differentiation capacity into ECs compared with control iPSCs. However, the Pi transport activity of IBGC patient-derived iPS-ECs was significantly decreased compared with that of control iPS-ECs without changing the gene expression of the other SLC 20 family members. We confirmed that SLC20A2 variants caused the loss of function of the Pi transport activity in both Flp-In CHO cells and disease-specific iPSCs. This is the first report to show an in vitro model of iPSCs in IBGC with patient-identified SLC20A2 variants. These useful tools will help in elucidating IBGC pathogenesis and can be used for screening drug candidates.

Inorganic phosphorus (Pi) in CSF is a biomarker for SLC20A2-associated idiopathic basal ganglia calcification (IBGC1).

INTRODUCTION: Idiopathic basal ganglia calcification (IBGC), also called Fahr's disease or recently primary familial brain calcification (PFBC), is characterized by abnormal deposits of minerals including calcium mainly and phosphate in the brain. Mutations in SLC20A2 (IBGC1 (merged with former IBGC2 and IBGC3)), which encodes PiT-2, a phosphate transporter, is the major cause of IBGC. Recently, Slc20a2-KO mice have been showed to have elevated levels of inorganic phosphorus (Pi) in cerebrospinal fluid (CSF); however, CSF Pi levels in patients with IBGC have not been fully examined. METHODS: We investigated the cases of 29 patients with IBGC including six patients with SLC20A2 mutation and three patients with PDGFB mutation, and 13 controls. The levels of sodium (Na), potassium (K), chloride (Cl), calcium (Ca), and Pi in sera and CSF were determined by potentiometry and colorimetry. Moreover, clinical manifestations were investigated in the IBGC patients with high Pi levels in CSF. RESULTS: The study revealed that the average level of Pi in the CSF of the total group of patients with IBGC is significantly higher than that of the control group, and the levels of Pi in CSF of the IBGC patients with SLC20A2 mutations are significantly higher than those of the IBGC patients with PDGFB mutations, the other IBGC patients and controls. CONCLUSION: Results of this study suggest that the levels of CSF Pi will be a good biomarker for IBGC1.

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.

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.

The effects of Brazilian green propolis that contains flavonols against mutant copper-zinc superoxide dismutase-mediated toxicity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. The purpose of this study was to clarify effects of brazilian green propolis and the active ingredient against ALS-associated mutant copper-zinc superoxide dismutase (SOD1)-mediated toxicity. Ethanol extract of brazilian green propolis (EBGP) protected N2a cells against mutant SOD1-induced neurotoxicity and reduced aggregated mutant SOD1 by induction of autophagy. Kaempferide and kaempferol, the active ingredients of EBGP, also inhibited mutant SOD1-induced cell death and reduced the intracellular mutant SOD1 aggregates. Both kaempferide and kaempferol significantly suppressed mutant SOD1-induced superoxide in mitochondria. Western blot analysis showed that kaempferol potentially induced autophagy via the AMP-activated protein kinase (AMPK) - the mammalian target of rapamycin (mTOR) pathway. These results suggest that EBGP containing the active ingredient against mutant SOD1-mediated toxicity is a promising medicine or health food for prevention and treatment of ALS.

The type III transporters (PiT-1 and PiT-2) are the major sodium-dependent phosphate transporters in the mice and human brains.

PiT-1/SLC20A1 and PiT-2/SLC20A2 are members of the mammalian type-III inorganic phosphate (Pi) transporters encoded by the SLC20 genes. The broad distribution of SLC20A1 and SLC20A2 mRNAs in mammalian tissues is compatible with housekeeping maintenance of intracellular Pi homeostasis by transporting Pi from intrastitial fluid for normal cellular functions. Recently, mutations of SLC20A2 have been found in patients with idiopathic basal ganglia calcification (IBGC), also known as Fahr's disease. However, the localization of PiT-1 and PiT-2 in the normal brain has not been clarified yet. The aim of this study was to reveal the distribution of PiT-1 and PiT-2 in the mouse and human brains. As results, gene expressing analysis showed that SLC20A1 and SLC20A2 mRNAs were widely expressed throughout the mouse and human brains, although other Pi transporters encoded by SLC17 and SLC34 mRNAs were hardly detected. The region of cerebellum contained a higher level of SLC20A1 and SLC20A2 mRNAs than the other brain regions. Additionally, the cerebellum in the mouse brain contained higher levels of PiT-1 and PiT-2 than those in the other regions in the brain, respectively. The immunohistochemical studies showed that PiT-1 was recognized in neuron, astrocytes and vascular endothelial cells. Similarly to PiT-1 immunopositivity, PiT-2 was clearly recognized in these cells. These results suggest that SLC20 family plays a pivotal role in the maintenance of cellular Pi homeostasis particularly in the brain. The viewpoint is compatible with the finding that calcification in IBGC is recognized only in the brain. This provides us with a novel viewpoint to understand the basic pathophysiology of IBGC through type-III Pi transporters.

Characterization of canine dental pulp cells and their neuroregenerative potential.

Dental pulp cells (DPCs) of various species have been studied for their potentials of differentiation into functional neurons and secretion of neurotrophic factors. In canine, DPCs have only been studied for cell surface markers and differentiation, but there is little direct evidence for therapeutic potentials for neurological disorders. The present study aimed to further characterize canine DPCs (cDPCs), particularly focusing on their neuroregenerative potentials. It was also reported that superparamagnetic iron oxide (SPIO) particles were useful for labeling of MSCs and tracking with magnetic resonance imaging (MRI). Our data suggested that cDPCs hold higher proliferation capacity than bone marrow stromal cells, the other type of mesenchymal stem cells which have been the target of intensive research. Canine DPCs constitutively expressed neural markers, suggesting a close relationship to the nervous system in their developmental origin. Canine DPCs promoted neuritogenesis of PC12 cells, most likely through secretion of neurotrophic factors. Furthermore, SPIO nanoparticles could be effectively transported to cDPCs without significant cytotoxicity and unfavorable effects on neuritogenesis. SPIO-labeled cDPCs embedded in agarose spinal cord phantoms were successfully visualized with a magnetic resonance imaging arousing a hope for noninvasive cell tracking in transplantation studies.

Conditioned medium of dental pulp cells stimulated by Chinese propolis show neuroprotection and neurite extension in vitro.

The purpose of this study was to clarify the effect of Chinese propolis on the expression level of neurotrophic factors in dental pulp cells (DPCs). We also investigated that the effects of the conditioned medium (CM) of DPCs stimulated by the propolis against oxidative and endoplasmic reticulum (ER) stresses in human neuroblastoma SH-SY5Y cells, and on neurite extensions in rat adrenal pheochromocytoma PC12 cells. To investigate the effect of the propolis on the levels of neurotrophic factors in DPCs, we performed a qRT-PCR experiment. As results, NGF, but not BDNF and NT-3, in DPCs was significantly elevated by the propolis in a concentration-dependent manner. H2O2-induced cell death was significantly inhibited by the treatment with the CM of DPCs. In addition, the treatment with the propolis-stimulated CM of DPCs had a more protective effect than that with the CM of DPCs. We also examine the effect of the propolis-stimulated CM of DPCs against a tunicamycin-induced ER stress. The treatment with the propolis-stimulated CM as well as the CM of DPCs significantly inhibited tunicamycin-induced cell death. Moreover, the treatment with the propolis-stimulated CM of DPCs significantly induced neurite outgrowth from PC12 cells than that with the CM of DPCs. These results suggest that the CM of DPCs as well as DPCs will be an efficient source of new treatments for neurodegenerative diseases and that the propolis promote the advantage of the CM of DPCs via producing neurotrophic factors.