Elimination of Mutant mtDNA by an Optimized mpTALEN Restores Differentiation Capacities of Heteroplasmic MELAS-iPSCs.

Various mitochondrial diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with heteroplasmic mutations in mitochondrial DNA (mtDNA). Herein, we refined a previously generated G13513A mtDNA-targeted platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate mtDNA heteroplasmy in MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying codons in their expression vectors could suppress the reduction in the mtDNA copy number, which contributed to the rapid recovery of mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover, MELAS-iPSCs with a high proportion of G13513A mutant mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally, drug-inducible, myogenic differentiation 1 (MYOD)-transfected MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into myosin heavy chain-positive myocytes, with or without mutant mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling.

Author Correction: TALEN-mediated shift of mitochondrial DNA heteroplasmy in MELAS-iPSCs with m.13513 G>A mutation.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

TALEN-mediated shift of mitochondrial DNA heteroplasmy in MELAS-iPSCs with m.13513G>A mutation.

Induced pluripotent stem cells (iPSCs) are suitable for studying mitochondrial diseases caused by mitochondrial DNA (mtDNA) mutations. Here, we generated iPSCs from a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with the m.13513G>A mutation. The patient's dermal fibroblasts were reprogrammed, and we established two iPSC clones with and without mutant mtDNA. Furthermore, we tried to decrease mutant mtDNA level in iPSCs using transcription activator-like effector nucleases (TALENs). We originally engineered platinum TALENs, which were transported into mitochondria, recognized the mtDNA sequence including the m.13513 position, and preferentially cleaved G13513A mutant mtDNA (G13513A-mpTALEN). The m.13513G>A heteroplasmy level in MELAS-iPSCs was decreased in the short term by transduction of G13513A-mpTALEN. Our data demonstrate that this mtDNA-targeted nuclease would be a powerful tool for changing the heteroplasmy level in heteroplasmic iPSCs, which could contribute to elucidation of the pathological mechanisms of mitochondrial diseases caused by mtDNA mutations.

Patients that have Undergone Hemodialysis Exhibit Lower Amyloid Deposition in the Brain: Evidence Supporting a Therapeutic Strategy for Alzheimer's Disease by Removal of Blood Amyloid.

As a proof of concept that removal of blood amyloid-ß (Aß) can reduce Aß deposition in the brains of patients with Alzheimer's disease, cortices of patients who had undergone hemodialysis (HD), which removes Aß from the blood, were histochemically analyzed; postmortem brain sections were stained with anti-Aß antibodies. Brains from patients who had undergone HD had significantly fewer senile plaques than those of patient who had not undergone HD. This significant difference was also confirmed by silver staining. Our findings suggest that removal of blood Aß by hemodialysis results in lower accumulation of Aß in the brain.

Intravenous administration of bone marrow mononuclear cells alleviates hearing loss after transient cochlear ischemia through paracrine effects.

Bone marrow mononuclear cells (BMMCs) are known to enhance recovery from ischemic insults by secreting angiogenic factors and inducing the expression of angiogenic factors from host tissues. Therefore, the transplantation of BMMCs is considered a potential approach to promoting the repair of ischemic damaged organs. Here, we investigated the influence of BMMCs on progressive hair cell degeneration after transient cochlear ischemia in gerbils. Transient cochlear ischemia was produced by extracranial occlusion of the bilateral vertebral arteries immediately before their entry into the transverse foramen of the cervical vertebra. An intravenous injection of BMMCs prevented ischemia-induced hair cell degeneration and ameliorated hearing impairment. A tracking study showed that BMMCs injected into the femoral vein were limited in the spiral artery of the cochlea, suggesting that, although transplanted BMMCs were retained within the spiral ganglion area of the cochlea, they were neither transdifferentiated into cochlear cells nor fused with the injured hair cells and supporting cells in the organ of Corti to restore their functions. We also showed that the protein level of neurotrophin-3 and glial cell line-derived neurotrophic factor in the organ of Corti was upregulated after treatment with BMMCs. These results suggested that BMMCs have therapeutic potential possibly through paracrine effects. Thus, we propose the use of BMMCs as a potential new therapeutic strategy for hearing loss.

Modeling Alzheimer's disease with iPSCs reveals stress phenotypes associated with intracellular Aß and differential drug responsiveness.

Oligomeric forms of amyloid-ß peptide (Aß) are thought to play a pivotal role in the pathogenesis of Alzheimer's disease (AD), but the mechanism involved is still unclear. Here, we generated induced pluripotent stem cells (iPSCs) from familial and sporadic AD patients and differentiated them into neural cells. Aß oligomers accumulated in iPSC-derived neurons and astrocytes in cells from patients with a familial amyloid precursor protein (APP)-E693Δ mutation and sporadic AD, leading to endoplasmic reticulum (ER) and oxidative stress. The accumulated Aß oligomers were not proteolytically resistant, and docosahexaenoic acid (DHA) treatment alleviated the stress responses in the AD neural cells. Differential manifestation of ER stress and DHA responsiveness may help explain variable clinical results obtained with the use of DHA treatment and suggests that DHA may in fact be effective for a subset of patients. It also illustrates how patient-specific iPSCs can be useful for analyzing AD pathogenesis and evaluating drugs.

Targeted disruption of organic cation transporter 3 (Oct3) ameliorates ischemic brain damage through modulating histamine and regulatory T cells.

The organic cation transporters OCT1, 2, and 3 (SLC22A1-3) have been implicated in the elimination of biogenic amines such as histamine. Among them, OCT3 was identified as an uptake-2 transporter, responsible for clearance of histamine. Because increasing evidence suggests the involvement of histamine in cerebral ischemia, we investigated the effects of targeted disruption of organic cation transporter-3 (Oct3) on the severity of ischemic brain damage. Transient focal ischemia for 1 hour was induced by occlusion of the middle cerebral artery (MCA) of homozygous Oct3-deficient mice and their wild-type (Wt) littermates. Although targeted disruption of Oct3 did not affect physiological parameters after MCA occlusion, this disruption significantly increased histamine content in the ischemic cortex and significantly reduced the infarct volume after cerebral ischemia. Furthermore, targeted disruption of Oct3 prevented the reduction of regulatory T-cell proportion after cerebral ischemia while this disruption did not affect Th1 and Th2 cells proportions after ischemia. Since repeated administration of L-histidine (a precursor of histamine) to Wt mice also showed the same effects, our observations suggested that OCT3 is the molecule responsible for clearance of ischemia-induced histamine in the brain and targeted disruption of Oct3 ameliorated ischemic brain damage through an increase in regulatory T cells.

Risk of progression from mild memory impairment to clinically diagnosable Alzheimer's disease in a Japanese community (from the Nakayama Study).

BACKGROUND: Memory impairment has been proposed as the most common early sign of Alzheimer's disease (AD). The aims of this work were to evaluate the risk of progression from mild memory impairment/no dementia (MMI/ND) to clinically diagnosable AD in a community-based prospective cohort and to establish the risk factors for progression from MMI/ND to AD in the elderly. METHODS: Elderly subjects aged over 65 years were selected from the participants in the first Nakayama study. MMI/ND was defined as memory deficit on objective memory assessment, without dementia, impairment of general cognitive function, or disability in activities of daily living. A total of 104 MMI/ND subjects selected from 1242 community-dwellers were followed longitudinally for five years. RESULTS: During the five-year follow-up, 11 (10.6%) subjects were diagnosed with AD, five (4.8%) with vascular dementia (VaD), and six (5.8%) with dementia of other etiology. Logistic regression analysis revealed that diabetes mellitus (DM) and a family history of dementia (within third-degree relatives) were positively associated with progression to AD, while no factor was significantly associated with progression to VaD or all types of dementia. CONCLUSIONS: DM and a family history of dementia were significant risk factors for progression from MMI/ND to clinically diagnosable AD in the elderly in a Japanese community.

Reduced ischemic brain injury by partial rejuvenation of bone marrow cells in aged rats.

Circulating bone marrow-derived immature cells, including endothelial progenitor cells, have been implicated in homeostasis of the microvasculature. Decreased levels of circulating endothelial progenitor cells, associated with aging and/or cardiovascular risk factors, correlate with poor clinical outcomes in a range of cardiovascular diseases. Herein, we transplanted bone marrow cells from young stroke-prone spontaneously hypertensive rats (SHR-SP) into aged SHR-SP, the latter not exposed to radiation or chemotherapy. Analysis of recipient peripheral blood 28 days after transplantation revealed that 5% of circulating blood cells were of donor origin. Cerebral infarction was induced on day 30 posttransplantation. Animals transplanted with bone marrow from young SHR-SP displayed an increase in density of the microvasculature in the periinfarction zone, reduced ischemic brain damage and improved neurologic function. In vitro analysis revealed enhanced activation of endothelial nitric oxide synthase and reduced activation p38 microtubule-associated protein (MAP) kinase, the latter associated with endothelial apoptosis, in cultures exposed to bone marrow-derived mononuclear cells from young animals versus cells from aged counterparts. Our findings indicate that partial rejuvenation of bone marrow from aged rats with cells from young animals enhances the response to ischemic injury, potentially at the level of endothelial/vascular activation, providing insight into a novel approach ameliorate chronic vascular diseases.

Delayed neuronal cell death in brainstem after transient brainstem ischemia in gerbils.

BACKGROUND: Because of the lack of reproducible brainstem ischemia models in rodents, the temporal profile of ischemic lesions in the brainstem after transient brainstem ischemia has not been evaluated intensively. Previously, we produced a reproducible brainstem ischemia model of Mongolian gerbils. Here, we showed the temporal profile of ischemic lesions after transient brainstem ischemia. RESULTS: Brainstem ischemia was produced by occlusion of the bilateral vertebral arteries just before their entry into the transverse foramina of the cervical vertebrae of Mongolian gerbils. Animals were subjected to brainstem ischemia for 15 min, and then reperfused for 0 d (just after ischemia), 1 d, 3 d and 7 d (n = 4 in each group). Sham-operated animals (n = 4) were used as control. After deep anesthesia, the gerbils were perfused with fixative for immunohistochemical investigation. Ischemic lesions were detected by immunostaining for microtubule-associated protein 2 (MAP2). Just after 15-min brainstem ischemia, ischemic lesions were detected in the lateral vestibular nucleus and the ventral part of the spinal trigeminal nucleus, and these ischemic lesions disappeared one day after reperfusion in all animals examined. However, 3 days and 7 days after reperfusion, ischemic lesions appeared again and clusters of ionized calcium-binding adapter molecule-1(IBA-1)-positive cells were detected in the same areas in all animals. CONCLUSION: These results suggest that delayed neuronal cell death took place in the brainstem after transient brainstem ischemia in gerbils.

Conditional deletion of Stat3 promotes neurogenesis and inhibits astrogliogenesis in neural stem cells.

Although signal transducer and activator of transcription 3 (Stat3) plays crucial roles in the determination of neural stem cell (NSC) fate, Stat3 has multiple roles in NSC function. Moreover, Stat3 plays important roles in neuronal survival and tumorigenesis. To investigate the overall effects of Stat3 on NSC fate, NSC were isolated from Stat3(flox/flox) mouse embryos (E14-15d), in which both Stat3 alleles are flanked by LoxP sites. Isolated NSC was inoculated with an adenovirus vector expressing Cre recombinase (Ad.nCre) or a control adenovirus vector expressing beta-galactosidase (Ad.nLz). Three days later, quantitative real-time PCR (qPCR) analysis revealed that treatment with Ad.nCre eliminated stat3 mRNA expression in NSC. Promoter assay confirmed that overexpression of nCre inhibited transactivation of acute responsive element (APRE) and blocked Stat3 function in NSC. Moreover, Western blot analysis and immunocytochemical analysis revealed that elimination of Stat3 in NSC promoted neurogenesis and inhibited astrogliogenesis. In addition, we investigated the effects of Stat3 elimination in NSC on the mRNA expression of Notch family members and bHLH factors. Consequently, qPCR analysis showed that elimination of Stat3 in NSC promoted neurogenesis and inhibited astrogliogenesis through down-regulation of notch1, notch2 and hes5, but not hes1 mRNA expression.

Ischemic tolerance in the cochlea.

Ischemic tolerance in the cochlea was investigated in a gerbil model of cochlear ischemia. Transient cochlear ischemia was produced by extracranial occlusion of the bilateral vertebral arteries. The gerbils were divided into two groups; single ischemia group and double ischemia group. In the single ischemia group, animals were subjected to lethal cochlear ischemia for 15 min. In the double ischemia group, animals were subjected to sublethal cochlear ischemia for 2 min at 2 days before lethal ischemia for 15 min. Consequently, sublethal ischemia prevented lethal ischemia-induced hair cell degeneration and ameliorated hearing impairment, suggesting ischemic tolerance in the cochlea.

Up-regulation of syntaxin1 in ischemic cortex after permanent focal ischemia in rats.

Syntaxin1 and synaptotagmin are located in the pre-synaptic terminals and play central roles in Ca(2+)-triggered neurotransmitter release. Because excessive synaptic transmission has been implicated in neuronal cell death after ischemia, we investigated the effects of cerebral ischemia on the levels of these proteins using a rat permanent focal ischemia model. Western blot analysis revealed that the protein level of syntaxin1 was significantly up-regulated in the ischemic core cortex and peri-ischemic cortex at 1 day after ischemia, while the protein level of synaptotagmin was not. Immunohistochemical analysis revealed that the protein level of syntaxin1 was markedly up-regulated in the ischemic areas where immunoreaction for MAP2 was lost. Furthermore, we showed that resident microglial cells were quite vulnerable to ischemia. Our data provide novel insights into the molecular mechanism of cerebral ischemia at the pre-synaptic terminals.

Postischemic mild hypothermia alleviates hearing loss because of transient ischemia.

The effect of postischemic mild hypothermia on the inner ear has not been clarified. In this study, we investigated whether hypothermia after transient ischemia could prevent cochlear damage and its therapeutic time window. Mongolian gerbils were divided into six groups: a sham-operation group, a normothermia group, and four hypothermia groups in which hypothermia was induced 1-7, 1-4, 3-6, and 6-9 h after reperfusion. Animals subjected to postischemic mild hypothermia within 3 h after reperfusion had attenuated hearing loss and inner hair cell loss. The protective effect was greater when hypothermia was induced earlier and had a longer duration. This implies that mild hypothermia after ischemia could have therapeutic effects for inner ear ischemic damage.

Ramified microglial cells promote astrogliogenesis and maintenance of neural stem cells through activation of Stat3 function.

The differentiation and proliferation of neural stem cells (NSCs) are regulated by a combination of their intrinsic properties (e.g., transcription factors, epigenetic factors, and microRNA regulation) and cell-extrinsic properties from the microenvironment around NSC (e.g., cytokines, growth factors, and cell-cell contact). Recently, there has been a great interest in clarifying the mechanism of the influence of the microenvironment on NSCs, especially cell-cell contact between NSCs and other types of cells nearby. In this study, we investigated whether microglial (Mi) cells influence the fate of NSCs. Coculture study showed that ramified Mi cells promoted astrogliogenesis and maintenance of NSCs through their paracrine effects. This microglia-induced astrogliogenesis was inhibited by AG490 and by overexpression of the dominant-negative form of Stat3 and SOCS3. Promoter assay revealed transactivation of Stat3 function in NSCs by Mi cells. Gene expression study revealed that mRNA of Notch family members (notch1-3) and sox9 in NSCs was significantly upregulated by Mi cells, and this up-regulation was inhibited by AG490. These results demonstrated that ramified Mi cells promoted astrogliogenesis and maintenance of NSCs by activating Stat3 function and via notch and sox9 signaling pathways.

Intravenous infusion of dihydroginsenoside Rb1 prevents compressive spinal cord injury and ischemic brain damage through upregulation of VEGF and Bcl-XL.

Red ginseng root (Panax Ginseng CA Meyer) has been used clinically by many Asian people for thousands of years without any detrimental effects. One of the major components of Red ginseng root is ginsenoside Rb(1) (gRb1). Previously, we showed that intravenous infusion of gRb1 ameliorated ischemic brain damage through upregulation of an anti-apoptotic factor, Bcl-x(L) and that topical application of gRb1 to burn wound lesion facilitated wound healing through upregulation of vascular endothelial growth factor (VEGF). In the present study, we produced dihydroginsenoside Rb1 (dgRb1), a stable chemical derivative of gRb1, and showed that intravenous infusion of dgRb1 improved spinal cord injury (SCI) as well as ischemic brain damage. As we expected, the effective dose of dgRb1 was ten times lower than that of gRb1. Intravenous infusion of dgRb1 at this effective dose did not affect brain temperature, blood pressure or cerebral blood flow, suggesting that dgRb1 rescued damaged neurons without affecting systemic parameters. In subsequent in vitro studies that focused on dgRb1-induced expression of gene products responsible for neuronal death or survival, we showed that dgRb1 could upregulate the expression of not only Bcl-x(L), but also a potent angiogenic and neurotrophic factor, VEGF. We also showed that dgRb1-induced expression of bcl-x(L) and VEGF mRNA was HRE (hypoxia response element) and STRE (signal transducers and activators of transcription 5 (Stat5) response element) dependent, respectively.

Ginsenoside Rb1 protects against damage to the spiral ganglion cells after cochlear ischemia.

The effects of transient cochlear ischemia on spiral ganglion cells (SGCs) were studied in Mongolian gerbils. Ischemic insult was induced by occluding the bilateral vertebral arteries of gerbils for 15min. Seven days after ischemia, the percentage of SGCs decreased to 67.5% from the preischemic baseline in the basal turn. Evaluation with immunohistochemical staining showed TUNEL-positive reactions in the SGCs with fragmented nuclei. In addition, we investigated the protective effects of ginsenoside Rb1 (gRb1) against ischemic injury to SGCs. Seven days after ischemia, the auditory brainstem response threshold shift was significantly reduced and the percentage of SGCs decreased to 90.2% from the preischemic baseline in the basal turn in the gRb1-treated group. These findings suggest that gRb1 prevented hearing loss caused by ischemic injury to SGCs in Mongolian gerbils.

Overexpression of SOCS3 inhibits astrogliogenesis and promotes maintenance of neural stem cells.

To investigate the effects of suppressors of cytokine signaling 3 (SOCS3) on neural stem cell fate, stem cells were infected with an adenoviral vector expressing SOCS3. Three days later, western blot analysis and immunocytochemical analysis revealed that the protein level of MAP2 and the number of MAP2-positive cells were significantly increased in SOCS3-transfected cells, whereas the protein level of GFAP and the number of GFAP-positive cells were significantly decreased. Furthermore, promoter assay revealed a significant reduction in the transcriptional level of signal transducer and activator of transcription 3 (Stat3) in the transfected cells. In addition, the mRNA levels of Notch family member (notch1) and inhibitory basic helix-loop-helix (bHLH) factors (hes5 and id3) were significantly up-regulated 1 day after overexpression of SOCS3. Three days after transfection, the mRNA level of hes5 was significantly decreased, whereas that of notch1 was still up-regulated. Moreover, all of SOCS3-positive cells expressed Nestin protein but did not express MAP2 or GFAP proteins. These data indicate that overexpression of SOCS3 induced neurogenesis and inhibited astrogliogenesis in neural stem cells. Our data also show that SOCS3 promoted maintenance of neural stem cells.

Prevention of ischemic neuronal death by intravenous infusion of a ginseng saponin, ginsenoside Rb(1), that upregulates Bcl-x(L) expression.

Almost all agents that exhibit neuroprotection when administered into the cerebral ventricles are ineffective or much less effective in rescuing damaged neurons when infused into the blood stream. Search for an intravenously infusible drug with a potent neuroprotective action is essential for the treatment of millions of patients suffering from acute brain diseases. Here, we report that postischemic intravenous infusion of a ginseng saponin, ginsenoside Rb(1) (gRb(1)) (C(54)H(92)O(23), molecular weight 1109.46) to stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery distal to the striate branches significantly ameliorated ischemia-induced place navigation disability and caused an approximately 50% decrease in the volume of the cortical infarct lesion in comparison with vehicle-infused ischemic controls. In subsequent studies that focused on gRb(1)-induced expression of gene products responsible for neuronal death or survival, we showed that gRb(1) stimulated the expression of the mitochondrion-associated antiapoptotic factor Bcl-x(L) in vitro and in vivo. Moreover, we revealed that a Stat5 responsive element in the bcl-x promoter became active in response to gRb(1) treatment. Ginsenoside Rb(1) appears to be a promising agent not only for the treatment of cerebral stroke, but also for the treatment of other diseases involving activation of mitochondrial cell death signaling.

Neural stem cells suppress the hearing threshold shift caused by cochlear ischemia.

Neural stem cells are multipotent progenitor cells that show self-renewal activity. In this study, we assessed the use of neural stem cells for ameliorating ischemia-reperfusion injury of the gerbil cochlea. Neural stem cells were injected into one inner ear through the round window 1 day after ischemic insult. Immunostaining for nestin showed that the distribution of neural stem cells was concentrated within the organ of Corti. Seven days after ischemia, the injury-induced auditory brainstem response threshold shift and progressive inner hair cell damage were markedly less on the neural stem cell-transplanted side. These results suggest that the transplantation of neural stem cells is therapeutically useful for preventing damage to hair cells that occurs after transient ischemia of the cochlea.