Naoko Kuzumaki

Mitochondrial dysfunction associated with increased oxidative stress and α-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue

BackgroundParkinson’s disease (PD) is a neurodegenerative disease characterized by selective degeneration of dopaminergic neurons in the substantia nigra (SN). The familial form of PD, PARK2, is caused by mutations in the parkin gene. parkin-knockout mouse models show some abnormalities, but they do not fully recapitulate the pathophysiology of human PARK2.ResultsHere, we generated induced pluripotent stem cells (iPSCs) from two PARK2 patients. PARK2 iPSC-derived neurons showed increased oxidative stress and enhanced activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. iPSC-derived neurons, but not fibroblasts or iPSCs, exhibited abnormal mitochondrial morphology and impaired mitochondrial homeostasis. Although PARK2 patients rarely exhibit Lewy body (LB) formation with an accumulation of α-synuclein, α-synuclein accumulation was observed in the postmortem brain of one of the donor patients. This accumulation was also seen in the iPSC-derived neurons in the same patient.ConclusionsThus, pathogenic changes in the brain of a PARK2 patient were recapitulated using iPSC technology. These novel findings reveal mechanistic insights into the onset of PARK2 and identify novel targets for drug screening and potential modified therapies for PD.

Epigenetic transcriptional activation of monocyte chemotactic protein 3 contributes to long-lasting neuropathic pain

A multiplex analysis for profiling the expression of candidate genes along with epigenetic modification may lead to a better understanding of the complex machinery of neuropathic pain. In the present study, we found that partial sciatic nerve ligation most remarkably increased the expression of monocyte chemotactic protein 3 (MCP-3, known as CCL7) a total of 33 541 genes in the spinal cord, which lasted for 4 weeks. This increase in MCP-3 gene transcription was accompanied by the decreased trimethylation of histone H3 at Lys27 at the MCP-3 promoter. The increased MCP-3 expression associated with its epigenetic modification observed in the spinal cord was almost abolished in interleukin 6 knockout mice with partial sciatic nerve ligation. Consistent with these findings, a single intrathecal injection of recombinant proteins of interleukin 6 significantly increased MCP-3 messenger RNA with a decrease in the level of Lys27 trimethylation of histone H3 at the MCP-3 promoter in the spinal cord of mice. Furthermore, deletion of the C-C chemokine receptor type 2 (CCR2) gene, which encodes a receptor for MCP-3, failed to affect the acceleration of MCP-3 expression in the spinal cord after partial sciatic nerve ligation. A robust increase in MCP-3 protein, which lasted for up to 2 weeks after surgery, in the dorsal horn of the spinal cord of mice with partial sciatic nerve ligation was seen mostly in astrocytes, but not microglia or neurons. On the other hand, the increases in both microglia and astrocytes in the spinal cord by partial sciatic nerve ligation were mostly abolished in interleukin 6 knockout mice. Moreover, this increase in microglia was almost abolished by CCR2 gene deletion, whereas the increase in astrocytes was not affected in nerve-ligated mice that lacked the CCR2 gene. We also found that either in vivo or in vitro treatment with MCP-3 caused robust microglia activation. Under these conditions, intrathecal administration of MCP-3 antibody suppressed the increase in microglia within the mouse spinal cord and neuropathic pain-like behaviours after nerve injury. With the use of a functional magnetic resonance imaging analysis, we demonstrated that a single intrathecal injection of MCP-3 induced dramatic increases in signal intensity in pain-related brain regions. These findings suggest that increased MCP-3 expression associated with interleukin 6 dependent epigenetic modification at the MCP-3 promoter after nerve injury, mostly in spinal astrocytes, may serve to facilitate astrocyte-microglia interaction in the spinal cord and could play a critical role in the neuropathic pain-like state.

Change in MicroRNAs Associated with Neuronal Adaptive Responses in the Nucleus Accumbens under Neuropathic Pain

Neuropathic pain is the most difficult type of pain to control, and patients lose their motivation for the purposive pursuit with a decrease in their quality of life. Using a functional magnetic resonance imaging analysis, we demonstrated that blood oxygenation level-dependent signal intensity was increased in the ipsilateral nucleus accumbens (N.Acc.) following peripheral nerve injury. microRNAs are small, noncoding RNA molecules that direct the post-transcriptional suppression of gene expression, and play an important role in regulating synaptic plasticity. In this study, we found that sciatic nerve ligation induced a drastic decrease in the expression of miR200b and miR429 in N.Acc. neurons. The expression of DNA methyltransferase 3a (DNMT3a), which is the one of the predicted targets of miR200b/429, was significantly increased in the limbic forebrain including N.Acc. at 7 d after sciatic nerve ligation. Double-immunolabeling with antibodies specific to DNMT3a and NR1 showed that DNMT3a-immunoreactivity in the N.Acc. was located in NR1-labeled neurons, indicating that increased DNMT3a proteins were dominantly expressed in postsynaptic neurons in the N.Acc. area under a neuropathic pain-like state. The results of these analyses provide new insight into an epigenetic modification that is accompanied by a dramatic decrease in miR200b and miR429 along with the dysfunction of “mesolimbic motivation/valuation circuitry” under a neuropathic pain-like state. These phenomena may result in an increase in DNMT3a in neurons of the N.Acc. under neuropathic pain, which leads to the long-term transcription-silencing of several genes.

Astrocytic activation in the anterior cingulate cortex is critical for sleep disorder under neuropathic pain

Insomnia, depression, and anxiety disorder are common problems for people with neuropathic pain. In this study, mild noxious heat stimuli increased the duration and number of spontaneous pain‐like behaviors in sciatic nerve‐ligated mice. We used functional magnetic resonance imaging to visualize the increased blood oxygenation level‐dependent signal intensity in the anterior cingulate cortex (ACC) of mice with sciatic nerve ligation under mild noxious stimuli. Such stimuli significantly increased the release of glutamate in the ACC of nerve‐ligated mice. In addition, sciatic nerve ligation and mild noxious stimuli changed the morphology of astrocytes in the ACC. Treatment of cortical astrocytes with glutamate caused astrocytic activation, as detected by a stellate morphology. Furthermore, glutamate induced the translocation of GAT‐3 to astrocyte cell membranes using primary cultured glial cells from the mouse cortex. Moreover, the GABA level at the synaptic cleft in the ACC of nerve‐ligated mice was significantly decreased exposure to mild noxious stimuli. Finally, we investigated whether astrocytic activation in the ACC could directly mediate sleep disorder. With the optogenetic tool channel rhodopsin‐2 (ChR2), we demonstrated that selective photostimulation of these astrocytes in vivo triggered sleep disturbance. Taken together, these results suggest that neuropathic pain‐like stimuli activated astrocytes in the ACC and decreased the extracellular concentration of GABA via an increase in the release of glutamate. Furthermore, these findings provide novel evidence that astrocytic activation in the ACC can mimic sleep disturbance in mice. Synapse 68:235–247, 2014.

Multiple Analyses of G-Protein Coupled Receptor (GPCR) Expression in the Development of Gefitinib-Resistance in Transforming Non-Small-Cell Lung Cancer

There is increasing evidence that functional crosstalk between GPCRs and EGFR contributes to the progression of colon, lung, breast, ovarian, prostate and head and neck tumors. In this study, we performed multiple analyses of GPCR expression in a gefitinib-resistant non-small cell lung cancer (NSCLC) cell line, H1975, which harbors an L858R/T790M mutation. To determine the expression profile of mRNAs encoding 384 GPCRs in normal human lung fibroblast (NHLF) and H1975 cells, a GPCR-specific microarray analysis was performed. A heat-map of the microarray revealed considerable differences in the expression of GPCRs between NHLF and H1975 cells. From the GPCR expression list, we selected some GPCR agonists/antagonist to investigate whether the respective ligands could affect the growth of H1975 cells. Among them, treatment with either a selective antagonist of adenosine A2a receptors, which were highly expressed in H1975 cell and another gefitinib-resistant NSCLC cells, HCC827GR cells or “small interfering RNA” (siRNA) targeting adenosine A2a receptors produced a significant decrease in cell viability of both H1975 and HCC827GR cells. Among up-regulated GPCRs in H1975 cells, Gs-, Gi- and Gq-coupled GPCRs were expressed almost equally. Among down-regulated GPCRs, Gi-coupled GPCRs were dominantly expressed in H1975 cells. The present results suggest that multilayered crosstalk between GPCRs and EGFR may play an important role in orchestrating downstream signaling molecules that are implicated in the development of gefitinib-resistant NSCLC.

Tumour resistance in induced pluripotent stem cells derived from naked mole-rats

The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype—ARF suppression-induced senescence (ASIS)—that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR.

Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling

Summary Modeling of neurological diseases using induced pluripotent stem cells (iPSCs) derived from the somatic cells of patients has provided a means of elucidating pathogenic mechanisms and performing drug screening. T cells are an ideal source of patient-specific iPSCs because they can be easily obtained from samples. Recent studies indicated that iPSCs retain an epigenetic memory relating to their cell of origin that restricts their differentiation potential. The classical method of differentiation via embryoid body formation was not suitable for T cell-derived iPSCs (TiPSCs). We developed a neurosphere-based robust differentiation protocol, which enabled TiPSCs to differentiate into functional neurons, despite differences in global gene expression between TiPSCs and adult human dermal fibroblast-derived iPSCs. Furthermore, neurons derived from TiPSCs generated from a juvenile patient with Parkinsons disease exhibited several Parkinsons disease phenotypes. Therefore, we conclude that TiPSCs are a useful tool for modeling neurological diseases.

Changes in circadian rhythm for mRNA expression of melatonin 1A and 1B receptors in the hypothalamus under a neuropathic pain‐like state

Several clinical reports on neuropathic pain of various etiologies have shown that it significantly interferes with sleep. Inadequate sleep due to neuropathic pain may contribute to the stressful negative consequences of living with pain. It is generally recognized that melatonin (MT) system in the hypothalmus is crusial for circadian rhythm and sleep‐wake transition. However, little, if any, is known about whether neuropathic pain could affect the MT system associated with sleep disturbance. In this study, we investigated the possible changes in circadian rhythm for the expression of MT receptors, especially MT1A and MT1B receptors, in the hypothalamus of mice with sciatic nerve ligation. The samples for real‐time RT‐PCR assay were prepared at 8:00, 14:00, 20:00, and 2:00 on day 7 after sciatic nerve ligation or sham operation. The mRNA expression of MT1A and MT1B receptors at 2:00 in sciatic nerve‐ligated mice, which exhibited thermal hyperalgesia along with an increase in wakefulness and a decrease in nonrapid eye movement sleep, was significantly greater than those in sham‐operated mice, whereas the levels of both MT1A and MT1B receptors at 8:00 in sciatic nerve‐ligated mice were significantly lower than those in sham‐operated mice. These findings suggest that neuropathic pain‐like stimuli lead to sleep disturbance in parallel with changes in circadian rhythm for mRNA expression of MT 1A and 1B receptors in the hypothalamus of mice. Synapse 68:153–158, 2014.

Epigenetic modulation at the CCR2 gene correlates with the maintenance of behavioral sensitization to methamphetamine

The intermittent administration of methamphetamine produces behavioral sensitization to methamphetamine. In the limbic forebrain, mainly including the nucleus accumbens, of mice that had been intermittently treated with methamphetamine, we found a significant increase in mRNA of a chemokine, CCR2. This increase was accompanied by a significant increase in histone H3 lysine 4 (H3K4) trimethylation at its promoter. Interestingly, the maintenance of sensitization to methamphetamine‐induced hyperlocomotion was significantly decreased in CCR2 knockout mice. These findings suggest that increased CCR2 associated with epigenetic modification after the intermittent administration of methamphetamine may be associated with the maintenance of sensitization to methamphetamine‐induced hyperlocomotion.

Single-cell trajectory analysis of human homogenous neurons carrying a rare RELN variant

Reelin is a protein encoded by the RELN gene that controls neuronal migration in the developing brain. Human genetic studies suggest that rare RELN variants confer susceptibility to mental disorders such as schizophrenia. However, it remains unknown what effects rare RELN variants have on human neuronal cells. To this end, the analysis of human neuronal dynamics at the single-cell level is necessary. In this study, we generated human-induced pluripotent stem cells carrying a rare RELN variant (RELN-del) using targeted genome editing; cells were further differentiated into highly homogeneous dopaminergic neurons. Our results indicated that RELN-del triggered an impaired reelin signal and decreased the expression levels of genes relevant for cell movement in human neurons. Single-cell trajectory analysis revealed that control neurons possessed directional migration even in vitro, while RELN-del neurons demonstrated a wandering type of migration. We further confirmed these phenotypes in neurons derived from a patient carrying the congenital RELN-del. To our knowledge, this is the first report of the biological significance of a rare RELN variant in human neurons based on individual neuron dynamics. Collectively, our approach should be useful for studying reelin function and evaluating mental disorder susceptibility, focusing on individual human neuronal migration.