Generation of one induced pluripotent stem cell line JUCGRMi004-A from a Charcot-Marie-Tooth disease type 1A (CMT1A) patient with PMP22 duplication.

The CMT1A variant accounts for over 60% of cases of Charcot-Marie-Tooth disease (CMT), one of the most common human neuropathies. The cause of CMT1A has been identified as the duplication of PMP22, a myelin protein expressed in Schwann cells. Yet, the pathological mechanisms have not been elucidated, and no treatment is currently available. In our study, we established an iPS cell line from a CMT1A patient with PMP22 duplication. The generated iPSCs maintain pluripotency and in vitro differentiation potency.

Generation of hiPSCs (JUCGRMi003-A) from a patient with Parkinson's disease with PARK2 mutation.

PARK2 is the most common autosomal recessive form of Parkinson's disease and is caused by mutations in parkin that result in early-onset loss of dopaminergic neurons in the substantia nigra. In this study, we established an induced pluripotent stem cell (iPSC) line from a patient harboring a homozygous exon 3 deletion in PARK2. The established iPSCs showed pluripotency, the capacity to differentiate into the three germ layers, and normal karyotypes.

Modulation of lower limb muscle corticospinal excitability during various types of motor imagery.

Motor imagery (MI) is used for rehabilitation and sports training. Previous studies focusing on the upper limb have investigated the effects of MI on corticospinal excitability in the muscles involved in the imagined movement (i.e., the agonist muscles). The present study focused on several lower-limb movements and investigated the influences of MI on corticospinal excitability in the lower limb muscles. Twelve healthy individuals (ten male and two female individuals) participated in this study. Motor-evoked potentials (MEP) from the rectus femoris (RF), biceps femoris (BF), tibialis anterior (TA), and soleus (SOL) muscles were elicited through transcranial magnetic stimulation (TMS) to the primary motor cortex during MI of knee extension, knee flexion, ankle dorsiflexion, and ankle plantarflexion and at rest. The results showed that the RF MEPs were significantly increased during MI in knee extension, ankle dorsiflexion, and ankle plantarflexion but not in knee flexion, compared with those at rest. The TA MEPs were significantly increased during MI in knee extension and foot dorsiflexion, while MEPs were not significantly different during MI in knee flexion and foot dorsiflexion than those at rest. For the BF and SOL muscles, there was no significant MEP modulation in either MI. These results demonstrated that corticospinal excitability of the RF and TA muscles was facilitated during MI of movements in which they are active and during MI of lower-limb movements in which they are not involved. On the contrary, corticospinal excitability of the BF and SOL muscles was not facilitated by MI of lower-limb movements. These results suggest that facilitation of corticospinal excitability depends on the muscle and the type of lower-limb MI.

Generation of a control iPS cell line (JUCGRMi005-A) with no abnormalities in Parkinson's disease-related genes.

Appropriate control induced pluripotent stem cells (iPSCs) are essential for studying iPSCs derived from patients with Parkinson's disease (PD). In this study, we established an iPSC line from a healthy male donor. The iPSCs showed pluripotency, capacity to differentiate into three germ layers, and normal karyotypes. Additionally, we confirmed that the iPSC line did not exhibit any PD-related gene abnormalities. This iPSC line will be useful for PD research.

Generation of a control iPS cell line (JUCGRMi006-A) with no abnormalities in Parkinson's disease-related genes.

The appropriate control of induced pluripotent stem cells (iPSCs) is essential for studying iPSCs derived from patients with Parkinson's disease (PD). Here, we established an iPSC line from a healthy female donor. The iPSCs were pluripotent, could differentiate into three germ layers, and had normal karyotypes. We also confirmed that the iPSC line exhibited no PD-related gene abnormalities. This iPSC line will be useful for PD research.

Generation of three clones (JUCGRMi002-A, B, C) of induced pluripotent stem cells from a Parkinson's disease patient with SNCA duplication.

Parkinson's disease is the second most common neurodegenerative disorder and is pathologically characterized by synuclein-rich aggregations (Lewy bodies) in neurons. Multiplication of the synuclein gene (SNCA) increases the mRNA and protein levels of synuclein, resulting in autosomal dominant hereditary Parkinson's disease. In the present study, we established three isogenic induced pluripotent stem cells (iPSCs) from a patient harboring SNCA duplication, which showed pluripotency, three-germ layer differentiation capacity, and normal karyotypes.

Reduced ER-mitochondrial contact sites and mitochondrial Ca2+ flux in PRKN-mutant patient tyrosine hydroxylase reporter iPSC lines.

Endoplasmic reticulum-mitochondrial contact sites (ERMCS) play an important role in mitochondrial dynamics, calcium signaling, and autophagy. Disruption of the ERMCS has been linked to several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). However, the etiological role of ERMCS in these diseases remains unclear. We previously established tyrosine hydroxylase reporter (TH-GFP) iPSC lines from a PD patient with a PRKN mutation to perform correlative light-electron microscopy (CLEM) analysis and live cell imaging in GFP-expressing dopaminergic neurons. Here, we analyzed ERMCS in GFP-expressing PRKN-mutant dopaminergic neurons from patients using CLEM and a proximity ligation assay (PLA). The PLA showed that the ERMCS were significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control under normal conditions. The reduction of the ERMCS in PRKN-mutant patient dopaminergic neurons was further enhanced by treatment with a mitochondrial uncoupler. In addition, mitochondrial calcium imaging showed that mitochondrial Ca2+ flux was significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control. These results suggest a defect in calcium flux from ER to mitochondria is due to the decreased ERMCS in PRKN-mutant patient dopaminergic neurons. Our study of ERMCS using TH-GFP iPSC lines would contribute to further understanding of the mechanisms of dopaminergic neuron degeneration in patients with PRKN mutations.

Involvement of casein kinase 1 epsilon/delta (Csnk1e/d) in the pathogenesis of familial Parkinson's disease caused by CHCHD2.

Parkinson's disease (PD) is a common neurodegenerative disorder that results from the loss of dopaminergic neurons. Mutations in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) gene cause a familial form of PD with α-Synuclein aggregation, and we here identified the pathogenesis of the T61I mutation, the most common disease-causing mutation of CHCHD2. In Neuro2a cells, CHCHD2 is in mitochondria, whereas the T61I mutant (CHCHD2T61I ) is mislocalized in the cytosol. CHCHD2T61l then recruits casein kinase 1 epsilon/delta (Csnk1e/d), which phosphorylates neurofilament and α-Synuclein, forming cytosolic aggresomes. In vivo, both Chchd2T61I knock-in and transgenic mice display neurodegenerative phenotypes and aggresomes containing Chchd2T61I , Csnk1e/d, phospho-α-Synuclein, and phospho-neurofilament in their dopaminergic neurons. Similar aggresomes were observed in a postmortem PD patient brain and dopaminergic neurons generated from patient-derived iPS cells. Importantly, a Csnk1e/d inhibitor substantially suppressed the phosphorylation of neurofilament and α-Synuclein. The Csnk1e/d inhibitor also suppressed the cellular damage in CHCHD2T61I -expressing Neuro2a cells and dopaminergic neurons generated from patient-derived iPS cells and improved the neurodegenerative phenotypes of Chchd2T61I mutant mice. These results indicate that Csnk1e/d is involved in the pathogenesis of PD caused by the CHCHD2T61I mutation.

A defined method for differentiating human iPSCs into midbrain dopaminergic progenitors that safely restore motor deficits in Parkinson's disease.

Background: Parkinson's disease (PD) is a progressive neurodegenerative condition that primarily affects motor functions; it is caused by the loss of midbrain dopaminergic (mDA) neurons. The therapeutic effects of transplanting human-induced pluripotent stem cell (iPSC)-derived mDA neural progenitor cells in animal PD models are known and are being evaluated in an ongoing clinical trial. However, However, improvements in the safety and efficiency of differentiation-inducing methods are crucial for providing a larger scale of cell therapy studies. This study aimed to investigate the usefulness of dopaminergic progenitor cells derived from human iPSCs by our previously reported method, which promotes differentiation and neuronal maturation by treating iPSCs with three inhibitors at the start of induction. Methods: Healthy subject-derived iPS cells were induced into mDA progenitor cells by the CTraS-mediated method we previously reported, and their proprieties and dopaminergic differentiation efficiency were examined in vitro. Then, the induced mDA progenitors were transplanted into 6-hydroxydopamine-lesioned PD model mice, and their efficacy in improving motor function, cell viability, and differentiation ability in vivo was evaluated for 16 weeks. Results: Approximately ≥80% of cells induced by this method without sorting expressed mDA progenitor markers and differentiated primarily into A9 dopaminergic neurons in vitro. After transplantation in 6-hydroxydopamine-lesioned PD model mice, more than 90% of the engrafted cells differentiated into the lineage of mDA neurons, and approximately 15% developed into mature mDA neurons without tumour formation. The grafted PD model mice also demonstrated significantly improved motor functions. Conclusion: This study suggests that the differentiation protocol for the preparation of mDA progenitors is a promising option for cell therapy in patients with PD.

Autophagy promotes cell survival by maintaining NAD levels.

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction.

Generation of three hiPSC clones from a Parkinson's disease patient with a heterozygous variant of VPS35 p.D620N.

Approximately 10% of Parkinson's disease cases are familial and more than 20 disease-related genes have been identified. The VPS35 gene causes a rare type of Parkinson's disease called PARK17, which is inherited in an autosomal dominant manner. The VPS35 gene encodes a retromer complex, but the pathogenic mechanism involved in PARK17 is unknown. Here, we established three isogenic induced pluripotent stem cell (iPSC) lines from a patient harboring a heterozygous VPS35 c.1858G > A (p.D620N) variant. The derived iPSCs showed pluripotency, the capacity to differentiate into three germ layers, and normal karyotypes.

Methods to Induce Small-Scale Differentiation of iPS Cells into Dopaminergic Neurons and to Detect Disease Phenotypes.

Disease-specific induced pluripotent stem (iPS) cells are useful tools to analyze the pathology of neurodegenerative diseases. In this chapter, we describe a procedure to efficiently induce small-scale differentiation of patient iPS cells into midbrain dopaminergic neurons to detect cell death and mitochondrial clearance by using immunostaining. A combination of our method described here and an image analysis system, such as the IN Cell Analyzer, will enable the quantitative assessment of cell vulnerability and mitochondrial quality control abnormalities in cells derived from patients with Parkinson's disease; this set-up might be used to perform drug screening.

Differentiation of Midbrain Dopaminergic Neurons from Human iPS Cells.

Human-induced pluripotent stem (iPS) cells provide a powerful means for analyzing disease mechanisms and drug screening, especially for neurological diseases, considering the difficulty to obtain live pathological tissue. The midbrain dopaminergic neurons of the substantia nigra are mainly affected in Parkinson's disease, but it is impossible to obtain and analyze viable dopaminergic neurons from live patients. This problem can be overcome by the induction of dopaminergic neurons from human iPS cells. Here, we describe an efficient method for differentiating human iPS cells into midbrain dopaminergic neurons. This protocol holds merit for obtaining a deeper understanding of the disease and for developing novel treatments.

Uniaxially fixed mechanical boundary condition elicits cellular alignment in collagen matrix with induction of osteogenesis.

Osteocytes differentiated from osteoblasts play significant roles as mechanosensors in modulating the bone remodeling process. While the well-aligned osteocyte network along the trabeculae with slender cell processes perpendicular to the trabeculae surface is known to facilitate the sensing of mechanical stimuli by cells and the intracellular communication in the bone matrix, the mechanisms underlying osteocyte network formation remains unclear. Here, we developed a novel in vitro collagen matrix system exerting a uniaxially-fixed mechanical boundary condition on which mouse osteoblast-like MC3T3-E1 cells were subcultured, evoking cellular alignment along the uniaxial boundary condition. Using a myosin II inhibitor, blebbistatin, we showed that the intracellular tension via contraction of actin fibers contributed to the cellular alignment under the influence of isometric matrix condition along the uniaxially-fixed mechanical boundary condition. Furthermore, the cells actively migrated inside the collagen matrix and promoted the expression of osteoblast and osteocyte genes with their orientations aligned along the uniaxially-fixed boundary condition. Collectively, our results suggest that the intracellular tension of osteoblasts under a uniaxially-fixed mechanical boundary condition is one of the factors that determines the osteocyte alignment inside the bone matrix.

Establishment of an in vitro model for analyzing mitochondrial ultrastructure in PRKN-mutated patient iPSC-derived dopaminergic neurons.

Mitochondrial structural changes are associated with the regulation of mitochondrial function, apoptosis, and neurodegenerative diseases. PRKN is known to be involved with various mechanisms of mitochondrial quality control including mitochondrial structural changes. Parkinson's disease (PD) with PRKN mutations is characterized by the preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, which has been suggested to result from the accumulation of damaged mitochondria. However, ultrastructural changes of mitochondria specifically in dopaminergic neurons derived from iPSC have rarely been analyzed. The main reason for this would be that the dopaminergic neurons cannot be distinguished directly among a mixture of iPSC-derived differentiated cells under electron microscopy. To selectively label dopaminergic neurons and analyze mitochondrial morphology at the ultrastructural level, we generated control and PRKN-mutated patient tyrosine hydroxylase reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines. Correlative light-electron microscopy analysis and live cell imaging of GFP-expressing dopaminergic neurons indicated that iPSC-derived dopaminergic neurons had smaller and less functional mitochondria than those in non-dopaminergic neurons. Furthermore, the formation of spheroid-shaped mitochondria, which was induced in control dopaminergic neurons by a mitochondrial uncoupler, was inhibited in the PRKN-mutated dopaminergic neurons. These results indicate that our established TH-GFP iPSC lines are useful for characterizing mitochondrial morphology, such as spheroid-shaped mitochondria, in dopaminergic neurons among a mixture of various cell types. Our in vitro model would provide insights into the vulnerability of dopaminergic neurons and the processes leading to the preferential loss of dopaminergic neurons in patients with PRKN mutations.

Reduced astrocytic reactivity in human brains and midbrain organoids with PRKN mutations.

Parkin (encoded by PRKN) is a ubiquitin ligase that plays an important role in cellular mitochondrial quality control. Mutations in PRKN cause selective dopaminergic cell loss in the substantia nigra and are presumed to induce a decrease in mitochondrial function caused by the defective clearance of mitochondria. Several studies have demonstrated that parkin dysfunction causes mitochondrial injury and astrocytic dysfunction. Using immunohistochemical methods, we analyzed astrocytic changes in human brains from individuals with PRKN mutations. Few glial fibrillary acidic protein- and vimentin-positive astrocytes were observed in the substantia nigra in PRKN-mutated subjects compared with subjects with idiopathic Parkinson's disease. We also differentiated patient-specific induced pluripotent stem cells into midbrain organoids and confirmed decreased numbers of glial fibrillary acidic protein-positive astrocytes in PRKN-mutated organoids compared with age- and sex-matched controls. Our study reveals PRKN-mutation-induced astrocytic alteration and suggests the possibility of an astrocyte-related non-autonomous cell death mechanism for dopaminergic neurons in brains of PRKN-mutated patients.

Astrocytes Protect Human Dopaminergic Neurons from α-Synuclein Accumulation and Propagation.

The pathologic hallmark of Parkinson's disease is the accumulation of α-synuclein-containing Lewy bodies/neurites almost exclusively in neurons, and rarely in glial cells. However, emerging evidence suggests that glia such as astrocytes play an important role in the development of α-synuclein pathology. Using induced pluripotent stem-derived dopaminergic neurons and astrocytes from healthy subjects and patients carrying mutations in lysosomal ATP13A2, a monogenic form of synucleinopathy, we found that astrocytes rapidly internalized α-synuclein, and exhibited higher lysosomal degradation rates compared with neurons. Moreover, coculturing astrocytes and neurons led to decreased accumulation of α-synuclein in neurons and consequently diminished interneuronal transfer of α-synuclein. These protective functions of astrocytes were attenuated by ATP13A2 deficiency, suggesting that the loss of ATP13A2 function in astrocytes at least partially contributes to neuronal α-synuclein pathology. Together, our results highlight the importance of lysosomal function in astrocytes in the pathogenesis of synucleinopathies.SIGNIFICANCE STATEMENT While most neurodegenerative disorders are characterized by the accumulation of aggregated mutant proteins exclusively in neurons, the contribution of glial cells in this process remains poorly explored. Here, we demonstrate that astrocytes contribute to the removal of extracellular α-synuclein and that disruption of this pathway caused by mutations in the Parkinson's disease-linked gene ATP13A2 result in α-synuclein accumulation in human dopaminergic neurons. We found that astrocytes also protect neurons from α-synuclein propagation, whereas ATP13A2 deficiency in astrocytes compromises this protective function. These results highlight astrocyte-mediated α-synuclein clearance as a potential therapeutic target in disorders characterized by the accumulation of α-synuclein, including Parkinson's disease.

Perforated duodenal diverticulum successfully treated with a combination of surgical drainage and endoscopic nasobiliary and nasopancreatic drainage: a case report.

BACKGROUND: Perforation of a duodenal diverticulum is a rare complication that may become fatal with a delay in appropriate treatment. However, the optimal treatment for perforated duodenal diverticulum remains controversial, ranging from conservative therapy to surgery including pancreatoduodenectomy. CASE PRESENTATION: The patient was a 60-year-old woman with no particular medical history who visited our hospital with chief complaints of continuous fever and right dorsal pain. Upon arrival, she had tenderness in the right upper quadrant of the abdomen. Laboratory data showed the elevation of inflammatory markers. Computed tomography revealed free air with abscess formation around the duodenum, which was diagnosed as duodenal perforation with abdominal abscess. We decided on emergent surgery, and we identified the perforation site on the dorsal side of the second portion of the duodenum intraoperatively. However, the inflammation around the perforation site was severe, and it was difficult to perform primary closure or dissection of the perforated diverticulum. Therefore, we finished surgery by placing four indwelling intra-abdominal tubes. Since postoperative day (POD) 1, the elevation of inflammation markers appeared to be uncontrollable, owing to the leakage of bile and pancreatic juice. We decided to perform endoscopic retrograde cholangiopancreatography on POD 2, and inserted endoscopic nasobiliary drainage and nasopancreatic drainage tubes. The patient showed a good postoperative course and was discharged on POD 57. CONCLUSIONS: Endoscopic nasobiliary and nasopancreatic drainage in combination with surgical drainage may be an effective treatment for perforated duodenal diverticulum.

Identifying Therapeutic Agents for Amelioration of Mitochondrial Clearance Disorder in Neurons of Familial Parkinson Disease.

Parkinson disease (PD) is a neurodegenerative disorder caused by the progressive loss of midbrain dopaminergic neurons, and mitochondrial dysfunction is involved in its pathogenesis. This study aimed to establish an imaging-based, semi-automatic, high-throughput system for the quantitative detection of disease-specific phenotypes in dopaminergic neurons from induced pluripotent stem cells (iPSCs) derived from patients with familial PD having Parkin or PINK1 mutations, which exhibit abnormal mitochondrial homeostasis. The proposed system recapitulates the deficiency of mitochondrial clearance, ROS accumulation, and increasing apoptosis in these familial PD-derived neurons. We screened 320 compounds for their ability to ameliorate multiple phenotypes and identified four candidate drugs. Some of these drugs improved the locomotion defects and reduced ATP production caused by PINK1 inactivation in Drosophila and were effective for idiopathic PD-derived neurons with impaired mitochondrial clearance. Our findings suggest that the proposed high-throughput system has potential for identifying effective drugs for familial and idiopathic PD.