LIN28A enhances the therapeutic potential of cultured neural stem cells in a Parkinson's disease model.

The original properties of tissue-specific stem cells, regardless of their tissue origins, are inevitably altered during in vitro culturing, lessening the clinical and research utility of stem cell cultures. Specifically, neural stem cells derived from the ventral midbrain lose their dopamine neurogenic potential, ventral midbrain-specific phenotypes, and repair capacity during in vitro cell expansion, all of which are critical concerns in using the cultured neural stem cells in therapeutic approaches for Parkinson's disease. In this study, we observed that the culture-dependent changes of neural stem cells derived from the ventral midbrain coincided with loss of RNA-binding protein LIN28A expression. When LIN28A expression was forced and sustained during neural stem cell expansion using an inducible expression-vector system, loss of dopamine neurogenic potential and midbrain phenotypes after long-term culturing was blocked. Furthermore, dopamine neurons that differentiated from neural stem cells exhibited remarkable survival and resistance against toxic insults. The observed effects were not due to a direct action of LIN28A on the differentiated dopamine neurons, but rather its action on precursor neural stem cells as exogene expression was switched off in the differentiating/differentiated cultures. Remarkable and reproducible behavioural recovery was shown in all Parkinson's disease rats grafted with neural stem cells expanded with LIN28A expression, along with extensive engraftment of dopamine neurons expressing mature neuronal and midbrain-specific markers. These findings suggest that LIN28A expression during stem cell expansion could be used to prepare therapeutically competent donor cells.

Neural stem cells secrete factors facilitating brain regeneration upon constitutive Raf-Erk activation.

The intracellular Raf-Erk signaling pathway is activated during neural stem cell (NSC) proliferation, and neuronal and astrocytic differentiation. A key question is how this signal can evoke multiple and even opposing NSC behaviors. We show here, using a constitutively active Raf (ca-Raf), that Raf-Erk activation in NSCs induces neuronal differentiation in a cell-autonomous manner. By contrast, it causes NSC proliferation and the formation of astrocytes in an extrinsic autocrine/paracrine manner. Thus, treatment of NSCs with medium (CM) conditioned in ca-Raf-transduced NSCs (Raf-CM; RCM) became activated to form proliferating astrocytes resembling radial glial cells (RGCs) or adult-type NSCs. Infusion of Raf-CM into injured mouse brains caused expansion of the NSC population in the subventricular zone, followed by the formation of new neurons that migrated to the damaged site. Our study shows an example how molecular mechanisms dissecting NSC behaviors can be utilized to develop regenerative therapies in brain disorders.

Generation of Dopamine Neurons from Rodent Fibroblasts through the Expandable Neural Precursor Cell Stage.

Recent groundbreaking work has demonstrated that combined expression of the transcription factors Brn2, Ascl1, and Myt1L (BAM; also known as Wernig factors) convert mouse fibroblasts into postmitotic neuronal cells. However, questions remain regarding whether trans-conversion is achieved directly or involves an intermediary precursor stage. Trans-conversion toward expandable neural precursor cells (NPCs) is more useful than direct one-step neuron formation with respect to yielding a sufficient number of cells and the feasibility of manipulating NPC differentiation toward certain neuron subtypes. Here, we show that co-expression of Wernig factors and Bcl-xL induces fibroblast conversion into NPCs (induced NPCs (iNPCs)) that are highly expandable for >100 passages. Gene expression analyses showed that the iNPCs exhibited high expression of common NPC genes but not genes specific to defined embryonic brain regions. This finding indicated that a regional identity of iNPCs was not established. Upon induction, iNPCs predominantly differentiated into astrocytes. However, the differentiation potential was not fixed and could be efficiently manipulated into general or specific subtypes of neurons by expression of additional genes. Specifically, overexpression of Nurr1 and Foxa2, transcription factors specific for midbrain dopamine neuron development, drove iNPCs to yield mature midbrain dopamine neurons equipped with presynaptic DA neuronal functions. We further assessed the therapeutic potential of iNPCs in Parkinson disease model rats.

Combined Nurr1 and Foxa2 roles in the therapy of Parkinson's disease.

Use of the physiological mechanisms promoting midbrain DA (mDA) neuron survival seems an appropriate option for developing treatments for Parkinson's disease (PD). mDA neurons are specifically marked by expression of the transcription factors Nurr1 and Foxa2. We show herein that Nurr1 and Foxa2 interact to protect mDA neurons against various toxic insults, but their expression is lost during aging and degenerative processes. In addition to their proposed cell-autonomous actions in mDA neurons, forced expression of these factors in neighboring glia synergistically protects degenerating mDA neurons in a paracrine mode. As a consequence of these bimodal actions, adeno-associated virus (AAV)-mediated gene delivery of Nurr1 and Foxa2 in a PD mouse model markedly protected mDA neurons and motor behaviors associated with nigrostriatal DA neurotransmission. The effects of the combined gene delivery were dramatic, highly reproducible, and sustained for at least 1 year, suggesting that expression of these factors is a promising approach in PD therapy.

Evaluating subjective domains of antipsychotic-induced adverse effects using heart rate variability.

AIMS: Antipsychotic-induced autonomic dysregulation may lead to a wide range of subjective side-effects in schizophrenia patients. Using heart rate variability (HRV) measures, we prospectively examined the relationship between subjective side-effects and cardiac autonomic regulation in unmedicated schizophrenia patients. METHODS: Forty-five unmedicated schizophrenia patients were assessed for antipsychotic-associated side-effects and HRV parameters at baseline and after 6 weeks of treatment. Psychiatric symptoms and subjective side-effects were assessed using the Positive and Negative Syndrome Scale (PANSS) and the Liverpool University Neuroleptic Side-effect Rating Scale (LUNSERS). RESULTS: Correlations between subjective adverse effects and HRV measures at baseline and at week 6 varied. Nonetheless, the changes in the psychic side-effects domain were significantly correlated with the changes in time-domain HRV measures and sample entropy (SampEn). In addition, the change in SampEn was significantly associated with that in the scores of extrapyramidal, anticholinergic, miscellaneous, and red herring domains as well as the mean total LUNSERS score. CONCLUSION: Baseline HRV measures may predict clinical response and adverse events associated with treatment adherence. Also, subjective side-effects may correspond well with the changes in neurocardiac dynamics, and the changes in SampEn may effectively reflect subjective discomfort in patients receiving antipsychotic treatment.

Epigenetic activation of the Foxa2 gene is required for maintaining the potential of neural precursor cells to differentiate into dopaminergic neurons after expansion.

Dysregulation of forkhead box protein A2 (Foxa2) expression in fetal ventral mesencephalon (VM)-derived neural precursor cells (NPCs) appears to be associated with the loss of their potential to differentiate into dopaminergic (DA) neurons after mitogenic expansion in vitro, hindering their efficient use as a transplantable cell source. Here, we report that epigenetic activation of Foxa2 in VM-derived NPCs by inducing histone hyperacetylation rescues the mitogenic-expansion-dependent decrease of differentiation potential to DA neurons. The silencing of Foxa2 gene expression after expansion is accompanied by repressive histone modifications, including hypoacetylation of histone H3 and H4 and trimethylation of H3K27 on the Foxa2 promoter, as well as on the global level. In addition, histone deacetylase 7 (HDAC7) is highly expressed during differentiation and recruited to the Foxa2 promoter. Induction of histone acetylation in VM-derived NPCs by either knockdown of HDAC7 or treatment with the HDAC inhibitor apicidin upregulates Foxa2 expression via hyperacetylation of H3 and a decrease in H3K27 trimethylation on the promoter regions, leading to the expression of DA neuron developmental genes and enhanced differentiation of DA neurons. These effects are antagonized by the expression of shRNAs specific for Foxa2 but enhanced by shRNA for HDAC7. Collectively, these findings indicate that loss of differentiation potential of expanded VM-derived NPCs is attributed to a decrease in Foxa2 expression and suggest that activation of the endogenous Foxa2 gene by epigenetic regulation might be an approach to enhance the generation of DA neurons.

Vitamin C facilitates dopamine neuron differentiation in fetal midbrain through TET1- and JMJD3-dependent epigenetic control manner.

Intracellular Vitamin C (VC) is maintained at high levels in the developing brain by the activity of sodium-dependent VC transporter 2 (Svct2), suggesting specific VC functions in brain development. A role of VC as a cofactor for Fe(II)-2-oxoglutarate-dependent dioxygenases has recently been suggested. We show that VC supplementation in neural stem cell cultures derived from embryonic midbrains greatly enhanced differentiation toward midbrain-type dopamine (mDA) neurons, the neuronal subtype associated with Parkinson's disease. VC induced gain of 5-hydroxymethylcytosine (5hmC) and loss of H3K27m3 in DA phenotype gene promoters, which are catalyzed by Tet1 and Jmjd3, respectively. Consequently, VC enhanced DA phenotype gene transcriptions in the progenitors by Nurr1, a transcription factor critical for mDA neuron development, to be more accessible to the gene promoters. Further mechanism studies including Tet1 and Jmjd3 knockdown/inhibition experiments revealed that both the 5hmC and H3K27m3 changes, specifically in the progenitor cells, are indispensible for the VC-mediated mDA neuron differentiation. We finally show that in Svct2 knockout mouse embryos, mDA neuron formation in the developing midbrain decreased along with the 5hmC/H3k27m3 changes. These findings together indicate an epigenetic role of VC in midbrain DA neuron development.

Doxycycline enhances survival and self-renewal of human pluripotent stem cells.

We here report that doxycycline, an antibacterial agent, exerts dramatic effects on human embryonic stem and induced pluripotent stem cells (hESC/iPSCs) survival and self-renewal. The survival-promoting effect was also manifest in cultures of neural stem cells (NSCs) derived from hESC/iPSCs. These doxycycline effects are not associated with its antibacterial action, but mediated by direct activation of a PI3K-AKT intracellular signal. These findings indicate doxycycline as a useful supplement for stem cell cultures, facilitating their growth and maintenance.

Foxa2 acts as a co-activator potentiating expression of the Nurr1-induced DA phenotype via epigenetic regulation.

Understanding how dopamine (DA) phenotypes are acquired in midbrain DA (mDA) neuron development is important for bioassays and cell replacement therapy for mDA neuron-associated disorders. Here, we demonstrate a feed-forward mechanism of mDA neuron development involving Nurr1 and Foxa2. Nurr1 acts as a transcription factor for DA phenotype gene expression. However, Nurr1-mediated DA gene expression was inactivated by forming a protein complex with CoREST, and then recruiting histone deacetylase 1 (Hdac1), an enzyme catalyzing histone deacetylation, to DA gene promoters. Co-expression of Nurr1 and Foxa2 was established in mDA neuron precursor cells by a positive cross-regulatory loop. In the presence of Foxa2, the Nurr1-CoREST interaction was diminished (by competitive formation of the Nurr1-Foxa2 activator complex), and CoREST-Hdac1 proteins were less enriched in DA gene promoters. Consequently, histone 3 acetylation (H3Ac), which is responsible for open chromatin structures, was strikingly increased at DA phenotype gene promoters. These data establish the interplay of Nurr1 and Foxa2 as the crucial determinant for DA phenotype acquisition during mDA neuron development.

Effects of clozapine on heart rate dynamics and their relationship with therapeutic response in treatment-resistant schizophrenia.

Previous studies have suggested the utility of nonlinear complexity measures of heart rate variability (HRV) in evaluating the regulatory capacity of the neuroautonomic system. The purpose of the present study was to investigate the effects of clozapine on the nonlinear complexity measures of HRV in patients with treatment-resistant schizophrenia to find novel electrophysiological markers that indicate response to clozapine treatment. Forty patients with treatment-resistant schizophrenia were evaluated during 8 weeks of clozapine monotherapy. For nonlinear complexity measures of HRV, the approximate entropy (ApEn) and sample entropy (SampEn) values were obtained. The response rate to clozapine was 37.5%. The results of multivariate analysis of covariance revealed that the ApEn and the SampEn values of HRV at week 8 were significantly higher in the responders than in the nonresponders. Repeated-measures analysis of covariance showed a significant group by time interaction effect in the ApEn and SampEn indices. The responder group showed an increasing pattern of change in these complexity measures after administration of clozapine, whereas the nonresponder group showed a decreasing pattern of change. These results suggest that the nonlinear dynamic complexity measures of HRV, which indicate the irregularity and complexity of the biosystem, may be useful in evaluating the therapeutic changes of neuroautonomic function in schizophrenia. The response to clozapine treatment is expected to be more favorable when the plasticity of the neuroautonomic system reflected in the nonlinear complexity measures is high.

Differential association of adiposity measures with heart rate variability measures in Koreans.

PURPOSE: Although obesity has been associated with imbalances in cardiac autonomic nervous system, it is unclear whether there are differential relationships between adiposity measures and heart rate variability (HRV) measures. We aimed to examine differences in the relationship between adiposity measures and HRV indices in a healthy Korean population. MATERIALS AND METHODS: In all, 1409 non-smokers (811 males, 598 females) without known histories of cardiovascular (CV), endocrine, or neurological diseases underwent adiposity measurements [(body mass index (BMI), percentage of body fat mass (PBF), and waist-to-hip ratio (WHR)], the HRV assessment (SDNN, RMSSD, LF, HF, LF/HF, and pNN50), and examination for CV risk factors (fasting glucose, LDL-cholesterol, HDL-cholesterol, triglycerides, hs-CRP, and blood pressure). RESULTS: Compared with BMI and PBF, WHR was more strongly correlated with each HRV index and more likely to predict decreased HRV (<15 percentile vs. ≥ 15 percentile of each HRV index) in ROC curves analysis. In linear regression analysis, all adiposity measures were inversely associated with each HRV measure before adjusting for age, gender, and CV risk factors (p<0.05). After adjusting for the covariates, WHR was inversely related to RMSSD, LF, and pNN50; PBF with RMSSD, HF, and pNN50; BMI with RMSSD (p<0.05). The inversed association between HRV indices and the gender-specific WHR tertile was significant for subjects with BMI ≥ 25 kg/m(2), but not for those with BMI <25 kg/m(2). CONCLUSION: WHR and PBF appear to be better indicators for low HRV than BMI, and the association between abdominal adiposity and HRV may be stronger in overweight subjects.

Patterns of cardiorespiratory coordination in young women with recurrent major depressive disorder treated with escitalopram or venlafaxine.

Evidence from previous studies suggests autonomic dysregulation in patients with major depressive disorder (MDD). Antidepressant treatment may also affect central autonomic function. We investigated whether the type of antidepressant might be associated with the pattern of cardiorespiratory coordination in non-depressed women with recurrent MDD. Resting electrocardiograms and respiratory signals were simultaneously recorded from 38 euthymic women with recurrent MDD who were treated with either escitalopram (n=19) or venlafaxine (n=19) monotherapy and from 38 healthy women. Linear measures of heart rate variability were extracted to assess cardiac autonomic control. Sample entropy (SampEn) was computed to assess the complexity of heart rate and respiratory signals, and cross-SampEn was calculated to measure the nonlinear interaction of both signals. Significant decreases in the cardiovagal tone and cardiorespiratory coupling of women with recurrent MDD receiving venlafaxine, and tendencies toward lower cardiovagal tone and cardiorespiratory coupling in women with recurrent MDD receiving escitalopram were observed when compared with healthy controls. Effect sizes for these differences were large between women receiving venlafaxine and healthy controls. We found a positive association between cardiorespiratory decoupling and venlafaxine dose. Norepinephrine-enhancement, within a therapeutic dose range, seems to be closely associated with decreased vagal tone and reduced nonlinear coupling between heart rate and respiration in euthymic women with recurrent MDD. However, the effects of serotonin enhancement on cardiovagal tone should be considered. Our results suggest that the pharmacodynamic properties of antidepressants may affect autonomic regulation of women with recurrent MDD even in euthymic state.

In vitro generation of mature dopamine neurons by decreasing and delaying the expression of exogenous Nurr1.

Neural stem/progenitor cell (NSC/NPC) cultures can be a source of dopamine (DA) neurons for experimental and transplantation purposes. Nurr1, a steroid receptor transcription factor, can overcome the limitations associated with differentiation of cultured NPCs into DA neurons. However, forced Nurr1 expression in NPC cultures generates non-neuronal and/or immature DA cells. We show here that the Nurr1 level and period of expression crucially affect the differentiation and maturation of Nurr1-induced DA neurons. Mature DA neurons were generated by manipulating Nurr1 expression patterns to resemble those in the developing midbrain.

An integrative assessment of the psychophysiologic alterations in young women with recurrent major depressive disorder.

OBJECTIVE: Alterations in neuroelectrical activities coincide with major depressive disorder (MDD). This study examines the pattern of cerebral activity and cardiac autonomic parameters of euthymic women with recurrent MDD. METHODS: Resting electroencephalograms and electrocardiograms were recorded from 20 women with MDD receiving escitalopram and 40 matched and healthy women. We computed frontal alpha asymmetry to evaluate the interhemispheric balance. Parameters of heart rate variability were extracted to assess cardiac autonomic control. Sample entropy was used to assess the complexity of neurocardiac dynamics. The relationship between cardiovagal activity and alpha electroencephalogram was examined with a coherence analysis. RESULTS: Multivariable analysis of variance revealed a differential pattern of psychophysiologic variables between MDD patients and controls (p = .03). MDD was associated with a tendency toward lower left frontal activity (-0.06 [standard deviation = 0.14] versus 0.04 [0.17] lnµV(2), p = .04). Discriminant analysis demonstrated more right frontal activation, a lower high-frequency heart rate power spectrum, and a higher ratio of the low- to high-frequency heart rate power spectrum in patients with MDD compared with controls. Residual depressive symptoms (r = -0.09 to 0.11, p = .63-.99) and escitalopram dosage (r = -0.09 to 0.28, p = .22-.84) were not correlated with autonomic measures. Coherence between normalized high-frequency component of the heart rate power spectrum and alpha power was not significant (F3, p = .27; F4, p = .16). CONCLUSIONS: Euthymic women with recurrent MDD have a distinctive psychophysiologic profile. This profile may reflect altered frontal activation and a reduced cardiovagal tone in depression.

Automatic detection of seizure termination during electroconvulsive therapy using sample entropy of the electroencephalogram.

Determining the exact duration of seizure activity is an important factor for predicting the efficacy of electroconvulsive therapy (ECT). In most cases, seizure duration is estimated manually by observing the electroencephalogram (EEG) waveform. In this article, we propose a method based on sample entropy (SampEn) that automatically detects the termination time of an ECT-induced seizure. SampEn decreases during seizure activity and has its smallest value at the boundary of seizure termination. SampEn reflects not only different states of regularity and complexity in the EEG but also changes in EEG amplitude before and after seizure activity. Using SampEn, we can more precisely determine seizure termination time and total seizure duration.

Prolonged membrane depolarization enhances midbrain dopamine neuron differentiation via epigenetic histone modifications.

Understanding midbrain dopamine (DA) neuron differentiation is of importance, because of physiological and clinical implications of this neuronal subtype. We show that prolonged membrane depolarization induced by KCl treatment promotes DA neuron differentiation from neural precursor cells (NPCs) derived from embryonic ventral midbrain (VM). Interestingly, the depolarization-induced increase of DA neuron yields was not abolished by L-type calcium channel blockers, along with no depolarization-mediated change of intracellular calcium level in the VM-derived NPCs (VM-NPCs), suggesting that the depolarization effect is due to a calcium-independent mechanism. Experiments with labeled DA neuron progenitors indicate that membrane depolarization acts at the differentiation fate determination stage and promotes the expression of DA phenotype genes (tyrosine hydroxylase [TH] and DA transporter [DAT]). Recruitment of Nurr1, a transcription factor crucial for midbrain DA neuron development, to the promoter of TH gene was enhanced by depolarization, along with increases of histone 3 acetylation (H3Ac) and trimethylation of histone3 on lysine 4 (H3K4m3), and decreases of H3K9m3 and H3K27m3 in the consensus Nurr1 binding regions of TH promoter. Depolarization stimuli on differentiating VM-NPCs also induced dissociation of methyl CpG binding protein 2 and related repressor complex molecules (repressor element-1 silencing transcription factor corepressor and histone deacetylase 1) from the CpG sites of TH and DAT promoters. Based on these findings, we suggest that membrane depolarization promotes DA neuron differentiation by opening chromatin structures surrounding DA phenotype genes and inhibiting the binding of corepressors, thus allowing transcriptional activators such as Nurr1 to access DA neuron differentiation gene promoter regions.

Breakdown of the intermediate-term fractal scaling exponent in sinus node dysfunction. New method for non-invasive evaluation of sinus node function.

BACKGROUND: The aim of the present study was to characterize the heart rate dynamics of sinus bradycardia (SB) from sinus node dysfunction (SND) using non-linear dynamical system analysis. No data are yet available on how the dynamics change in the presence of SND. METHODS AND RESULTS: Conventional time and frequency domain analysis, the short- (DFAα(1)) and intermediate-term fractal scaling exponent (DFAα(2)), approximate entropy (ApEn) and sample entropy (SampEn) were calculated in 60-min sinus RR interval data of SB from 24-h ambulatory electrocardiograms of 110 patients: 44 SND patients, 44 age-matched controls, and 22 younger controls. All of the time and frequency domain parameters, ApEn and SampEn, were significantly reduced in the age-matched control group, compared with the young control group. DFAα(1) and DFAα(2) increased with aging. Both the DFAα(1) and DFAα(2) of SND patients were paradoxically reduced, which was not appropriate for their age. Only the percentage of consecutive RR intervals with absolute differences >50ms (pNN(50)), low-frequency power, and DFAα(2) made a significant contribution to prediction of SND on logistic regression analysis. Among them, DFAα(2) was the most significant variable for prediction of SND (odds ratio, 0.927; 95% confidence interval: 0.888-0.969, P=0.001). DFAα(2) remained as a significant variable for prediction of SND, when compared with overall control patients, combining the 2 control groups. CONCLUSIONS: Inappropriate reduction of DFAα(2) is a robust measure and could be an adjunctive tool for improvement of diagnostic performance in detection of SND.

Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease.

Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell-based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.

Association of heart rate variability with the framingham risk score in healthy adults.

BACKGROUND: The aim of this study was to investigate the relationship between heart rate variability (HRV), the Framingham risk score (FRS), and the 10-year risk of coronary heart disease (CHD) development among Korean adults. METHODS: The subjects were 85 healthy Korean adults recruited from a health check-up center. The FRS and 10-year risk of CHD development were calculated. RESULTS: The FRS in men was inversely correlated with the standard deviation of all normal to normal RR-intervals (SDNN); the root mean square successive difference (RMSSD); the percentage of successive normal cardiac inter-beat intervals greater than 20 ms, 30 ms, and 50 ms (pNN20, pNN30, pNN50); the low frequency (LF); and the high frequency (HF) (P < 0.05). There was no significant relationship between the FRS and HRV in women. Overall, in the receiver operating characteristic (ROC) analysis, the RMSSD, HF, SDNN, LF, LF/HF ratio, and pNN30 predicted an increased 10-year CHD risk. After adjusting for sex and body mass index, those with greater than one standard deviation in the RMSSD, HF, and LF had a 52-59% reduction in their 10-year risk of CHD development ≥ 10%. CONCLUSION: This study therefore indicates that the HRV indices, particularly SDNN, RMSSD, pNN30, LF, and HF may be useful parameters for the assessment of CHD risk. Most notably, the usefulness of these HRV measures as indicators for CHD risk evaluation may be greater among men than among women.