PGC-1α4 Interacts with REST to Upregulate Neuronal Genes and Augment Energy Consumption in Developing Cardiomyocytes.

Transcriptional coactivator PGC-1α is a main regulator of cardiac energy metabolism. In addition to canonical PGC-1α1, other PGC-1α isoforms have been found to exert specific biological functions in a variety of tissues. We investigated the expression patterns and the biological effects of the non-canonical isoforms in the heart. We used RNA sequencing data to identify the expression patterns of PGC-1α isoforms in the heart. To evaluate the biological effects of the alternative isoform expression, we generated a transgenic mouse with cardiac-specific overexpression of PGC-1α4 and analysed the cardiac phenotype with a wide spectrum of physiological and biophysical tools. Our results show that non-canonical isoforms are expressed in the heart, and that the main variant PGC-1α4 is induced by ß-adrenergic signalling in adult cardiomyocytes. Cardiomyocyte specific PGC-1α4 overexpression in mice relieves the RE1-Silencing Transcription factor (REST)-mediated suppression of neuronal genes during foetal heart development. The resulting de-repression of REST target genes induces a cardiac phenotype with increased cellular energy consumption, resulting in postnatal dilated cardiomyopathy. These results propose a new concept for actions of the PGC-1α protein family where activation of the Pgc-1α gene, through its isoforms, induces a phenotype with concurrent supply and demand for cellular energy. These data highlight the biological roles of the different PGC-1α isoforms, which should be considered when future therapies are developed.

Varied Responses to a High m.3243A>G Mutation Load and Respiratory Chain Dysfunction in Patient-Derived Cardiomyocytes.

The m.3243A>G mutation in mitochondrial tRNA-Leu(UUR) is one of the most common pathogenic mitochondrial DNA mutations in humans. The clinical manifestations are highly heterogenous and the causes for the drastic clinical variability are unknown. Approximately one third of patients suffer from cardiac disease, which often increases mortality. Why only some patients develop cardiomyopathy is unknown. Here, we studied the molecular effects of a high m.3243A>G mutation load on cardiomyocyte functionality, using cells derived from induced pluripotent stem cells (iPSC-CM) of two different m.3243A>G patients, only one of them suffering from severe cardiomyopathy. While high mutation load impaired mitochondrial respiration in both patients' iPSC-CMs, the downstream consequences varied. mtDNA mutant cells from a patient with no clinical heart disease showed increased glucose metabolism and retained cellular ATP levels, whereas cells from the cardiac disease patient showed reduced ATP levels. In this patient, the mutations also affected intracellular calcium signaling, while this was not true in the other patient's cells. Our results reflect the clinical variability in mitochondrial disease patients and show that iPSC-CMs retain tissue specific features seen in patients.

PGC-1α deficiency reveals sex-specific links between cardiac energy metabolism and EC-coupling during development of heart failure in mice.

AIMS: Biological sex has fundamental effects on mammalian heart physiology and pathogenesis. While it has been established that female sex is a protective factor against most cardiovascular diseases (CVDs), this beneficial effect may involve pathways associated with cardiac energy metabolism. Our aim was to elucidate the role of transcriptional coactivator PGC-1α in sex dimorphism of heart failure (HF) development. METHODS AND RESULTS: Here, we show that mice deficient in cardiac expression of the peroxisome proliferator-activated receptor gamma (PPAR-γ) coactivator-1α (PGC-1α) develop dilated HF associated with changes in aerobic and anaerobic metabolism, calcium handling, cell structure, electrophysiology, as well as gene expression. These cardiac changes occur in both sexes, but female mice develop an earlier and more severe structural and functional phenotype associated with dyssynchronous local calcium release resulting from disruption of t-tubular structures of the cardiomyocytes. CONCLUSIONS: These data reveal that the integrity of the subcellular Ca2+ release and uptake machinery is dependent on energy metabolism and that female hearts are more prone to suffer from contractile dysfunction in conditions with compromised production of cellular energy. Furthermore, these findings suggest that PGC-1α is a central mediator of sex-specific differences in heart function and CVD susceptibility.

Pteridophyte distribution of the Urals and adjacent areas: a dataset.

BACKGROUND: Data on the species diversity and distribution of pteridophytes (lycophytes and ferns) in the Urals and adjacent areas are presented. The dataset includes 13,742 observations of two classes Lycopodiopsida and Polypodiopsida. In total, the dataset contains information on 16 families, 28 generas, 65 species, four subspecies and nine interspecies hybrids. All records are for lycophytes and ferns collected over 170 years between 1853 and 2021. The dataset presented is based on herbarium specimens, published data and field research conducted by the authors. This dataset is the first and important step towards generalising information on the current diversity and geographical distribution of pteridophytes in the Urals and adjacent areas. NEW INFORMATION: The dataset contains 13,742 records of 65 species of pteridophytes occurrences in the Urals and adjacent territories: Udmurt Republic (42,100 km2); Perm Krai (160,600 km2); Sverdlovsk Oblast (194,800 km2); Chelyabinsk Oblast (87,900 km2); Republic of Bashkortostan (143,600 km2); Tyumen Oblast (160,100 km2); Yamalo-Nenets Autonomous Okrug (769,300 km2); Khanty-Mansi Autonomous Okrug (534,800km2) and Kurgan Oblast (71,500 km2). Each record includes a geographical description of the place of discovery and habitat, year of discovery, author of the finding and determination, as well as a link to a literary source (if the data were published) or the place of storage of the herbarium specimen. The presented dataset supplements the information on the occurrence of pteridophytes in the Russian Federation as a whole and clarifies their distribution in the Urals.

Metabolic alterations in Parkinson's disease astrocytes.

In Parkinson`s disease (PD), the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta is associated with Lewy bodies arising from the accumulation of alpha-synuclein protein which leads ultimately to movement impairment. While PD has been considered a disease of the DA neurons, a glial contribution, in particular that of astrocytes, in PD pathogenesis is starting to be uncovered. Here, we report findings from astrocytes derived from induced pluripotent stem cells of LRRK2 G2019S mutant patients, with one patient also carrying a GBA N370S mutation, as well as healthy individuals. The PD patient astrocytes manifest the hallmarks of the disease pathology including increased expression of alpha-synuclein. This has detrimental consequences, resulting in altered metabolism, disturbed Ca2+ homeostasis and increased release of cytokines upon inflammatory stimulation. Furthermore, PD astroglial cells manifest increased levels of polyamines and polyamine precursors while lysophosphatidylethanolamine levels are decreased, both of these changes have been reported also in PD brain. Collectively, these data reveal an important role for astrocytes in PD pathology and highlight the potential of iPSC-derived cells in disease modeling and drug discovery.

Short high-fat diet interferes with the physiological maturation of the late adolescent mouse heart.

Dietary fats are essential for cardiac function. The metabolites of fats known as fatty acids provide most of the energy for cardiac tissue, serve as building blocks for membranes and regulate important signaling cascades. Despite their importance, excess fat intake can cause cardiac dysfunction. The detrimental effects of high-fat diet (HFD) on cardiac health are widely investigated in long-term studies but the short-term effects of fats have not been thoroughly studied. To elucidate the near-term effects of a HFD on the growth and maturation of late adolescent heart we subjected 11-week-old mice to an 8-week long HFD (42% of calories from fat, 42% from carbohydrate, n = 8) or chow diet (12% of calories from fat, 66% from carbohydrate, n = 7) and assessed their effects on the heart in vivo and in vitro. Our results showed that excessive fat feeding interferes with normal maturation of the heart indicated by the lack of increase in dimensions, volume, and stroke volume of the left ventricles of mice on high fat diet that were evident in mice on chow diet. In addition, differences in regional strain during the contraction cycle between mice on HFD and chow diet were seen. These changes were associated with reduced activity of the growth promoting PI3K-Akt1 signaling cascade and moderate changes in glucose metabolism without changes in calcium signaling. This study suggests that even a short period of HFD during late adolescence hinders cardiac maturation and causes physiological changes that may have an impact on the cardiac health in adulthood.

Correct expression and localization of collagen XIII are crucial for the normal formation and function of the neuromuscular system.

Transmembrane collagen XIII has been linked to maturation of the musculoskeletal system. Its absence in mice (Col13a1-/- ) results in impaired neuromuscular junction (NMJ) differentiation and function, while transgenic overexpression (Col13a1oe ) leads to abnormally high bone mass. Similarly, loss-of-function mutations in COL13A1 in humans produce muscle weakness, decreased motor synapse function and mild dysmorphic skeletal features. Here, analysis of the exogenous overexpression of collagen XIII in various muscles revealed highly increased transcript and protein levels, especially in the diaphragm. Unexpectedly, the main location of exogenous collagen XIII in the muscle was extrasynaptic, in fibroblast-like cells, while some motor synapses were devoid of collagen XIII, possibly due to a dominant negative effect. Concomitantly, phenotypical changes in the NMJs of the Col13a1oe mice partly resembled those previously observed in Col13a1-/- mice. Namely, the overall increase in collagen XIII expression in the muscle produced both pre- and postsynaptic abnormalities at the NMJ, especially in the diaphragm. We discovered delayed and compromised acetylcholine receptor (AChR) clustering, axonal neurofilament aggregation, patchy acetylcholine vesicle (AChV) accumulation, disrupted adhesion of the nerve and muscle, Schwann cell invagination and altered evoked synaptic function. Furthermore, the patterns of the nerve trunks and AChR clusters in the diaphragm were broader in the adult muscles, and already prenatally in the Col13a1oe mice, suggesting collagen XIII involvement in the development of the neuromuscular system. Overall, these results confirm the role of collagen XIII at the neuromuscular synapses and highlight the importance of its correct expression and localization for motor synapse formation and function.

Structural Immaturity of Human iPSC-Derived Cardiomyocytes: In Silico Investigation of Effects on Function and Disease Modeling.

Background: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising experimental tool for translational heart research and drug development. However, their usability as a human adult cardiomyocyte model is limited by their functional immaturity. Our aim is to analyse quantitatively those characteristics and how they differ from adult CMs. Methods and Results: We have developed a novel in silico model with all essential functional electrophysiology and calcium handling features of hiPSC-CMs. Importantly, the virtual cell recapitulates the immature intracellular ion dynamics that are characteristic for hiPSC-CMs, as quantified based our in vitro imaging data. The strong "calcium clock" is a source for a dual function of excitation-contraction coupling in hiPSC-CMs: action potential and calcium transient morphology vary substantially depending on the activation sequence of underlying ionic currents and fluxes that is altered in spontaneous vs. paced mode. Furthermore, parallel simulations with hiPSC-CM and adult cardiomyocyte models demonstrate the central differences. Results indicate that hiPSC-CMs translate poorly the disease specific phenotypes of Brugada syndrome, long QT Syndrome and catecholaminergic polymorphic ventricular tachycardia, showing less robustness and greater tendency for arrhythmic events than adult CMs. Based on a comparative sensitivity analysis, hiPSC-CMs share some features with adult CMs, but are still functionally closer to prenatal CMs than adult CMs. A database analysis of 3000 hiPSC-CM model variants suggests that hiPSC-CMs recapitulate poorly fundamental physiological properties of adult CMs. Single modifications do not appear to solve this problem, which is mostly contributed by the immaturity of intracellular calcium handling. Conclusion: Our data indicates that translation of findings from hiPSC-CMs to human disease should be made with great caution. Furthermore, we established a mathematical platform that can be used to improve the translation from hiPSC-CMs to human, and to quantitatively evaluate hiPSC-CMs development toward more general and valuable model for human cardiac diseases.

PSEN1 Mutant iPSC-Derived Model Reveals Severe Astrocyte Pathology in Alzheimer's Disease.

Alzheimer's disease (AD) is a common neurodegenerative disorder and the leading cause of cognitive impairment. Due to insufficient understanding of the disease mechanisms, there are no efficient therapies for AD. Most studies have focused on neuronal cells, but astrocytes have also been suggested to contribute to AD pathology. We describe here the generation of functional astrocytes from induced pluripotent stem cells (iPSCs) derived from AD patients with PSEN1 ΔE9 mutation, as well as healthy and gene-corrected isogenic controls. AD astrocytes manifest hallmarks of disease pathology, including increased ß-amyloid production, altered cytokine release, and dysregulated Ca2+ homeostasis. Furthermore, due to altered metabolism, AD astrocytes show increased oxidative stress and reduced lactate secretion, as well as compromised neuronal supportive function, as evidenced by altering Ca2+ transients in healthy neurons. Our results reveal an important role for astrocytes in AD pathology and highlight the strength of iPSC-derived models for brain diseases.

Aggravated Postinfarct Heart Failure in Type 2 Diabetes Is Associated with Impaired Mitophagy and Exaggerated Inflammasome Activation.

Type 2 diabetes mellitus (T2DM) is a major risk factor for heart disease. Mortality rates after myocardial infarction (MI) are significantly increased in T2DM patients because of dysfunctional left ventricle (LV). However, molecular pathways underlying accelerated heart failure (HF) after MI in T2DM remain unclear. We investigated the underlying mechanisms by inducing MI in a well-established model of T2DM and control mice. Cardiac imaging revealed a significantly decreased global left ventricular ejection fraction in parallel with increased mortality after MI in T2DM mice compared with control mice. Genome-wide mRNA sequencing, immunoblot, electron microscopy, together with immunofluorescence staining for LC3 and p62 indicated an impaired mitophagy in peri-infarct regions of LV in T2DM mice compared with control mice. Furthermore, defective mitophagy was associated with an increased release of mitochondrial DNA, resulting in Aim2 and NLRC4 inflammasome and caspase-I hyperactivation in cardiomyocytes and cardiac macrophages in peri-infarct regions of LV in T2DM mice. Consistent with inflammasome and caspase-I hyperactivation, cardiomyocyte death and IL-18 secretion were increased in T2DM mice. Our results indicate that T2DM aggravates HF after MI through defective mitophagy, associated exaggerated inflammasome activation, cell death, and IL-18 secretion, suggesting that restoring mitophagy and inhibiting inflammasome activation may serve as novel targets for the prevention and treatment of HF in T2DM.

Loss of Cln5 causes altered neurogenesis in a mouse model of a childhood neurodegenerative disorder.

Neural stem/progenitor cells (NPCs) generate new neurons in the brain throughout an individual's lifetime in an intricate process called neurogenesis. Neurogenic alterations are a common feature of several adult-onset neurodegenerative diseases. The neuronal ceroid lipofuscinoses (NCLs) are the most common group of inherited neurodegenerative diseases that mainly affect children. Pathological features of the NCLs include accumulation of lysosomal storage material, neuroinflammation and neuronal degeneration, yet the exact cause of this group of diseases remains poorly understood. The function of the CLN5 protein, causative of the CLN5 disease form of NCL, is unknown. In the present study, we sought to examine neurogenesis in the neurodegenerative disorder caused by loss of Cln5 Our findings demonstrate a newly identified crucial role for CLN5 in neurogenesis. We report for the first time that neurogenesis is increased in Cln5-deficient mice, which model the childhood neurodegenerative disorder caused by loss of Cln5 Our results demonstrate that, in Cln5 deficiency, proliferation of NPCs is increased, NPC migration is reduced and NPC differentiation towards the neuronal lineage is increased concomitantly with functional alterations in the NPCs. Moreover, the observed impairment in neurogenesis is correlated with increased expression of the pro-inflammatory cytokine IL-1ß. A full understanding of the pathological mechanisms that lead to disease and the function of the NCL proteins are critical for designing effective therapeutic approaches for this devastating neurodegenerative disorder.

Vascular Endothelial Growth Factor-B Induces a Distinct Electrophysiological Phenotype in Mouse Heart.

Vascular endothelial growth factor B (VEGF-B) is a potent mediator of vascular, metabolic, growth, and stress responses in the heart, but the effects on cardiac muscle and cardiomyocyte function are not known. The purpose of this study was to assess the effects of VEGF-B on the energy metabolism, contractile, and electrophysiological properties of mouse cardiac muscle and cardiac muscle cells. In vivo and ex vivo analysis of cardiac-specific VEGF-B TG mice indicated that the contractile function of the TG hearts was normal. Neither the oxidative metabolism of isolated TG cardiomyocytes nor their energy substrate preference showed any difference to WT cardiomyocytes. Similarly, myocyte Ca2+ signaling showed only minor changes compared to WT myocytes. However, VEGF-B overexpression induced a distinct electrophysiological phenotype characterized by ECG changes such as an increase in QRSp time and decreases in S and R amplitudes. At the level of isolated TG cardiomyocytes, these changes were accompanied with decreased action potential upstroke velocity and increased duration (APD60-70). These changes were partly caused by downregulation of sodium current (INa) due to reduced expression of Nav1.5. Furthermore, TG myocytes had alterations in voltage-gated K+ currents, namely decreased density of transient outward current (Ito) and total K+ current (Ipeak). At the level of transcription, these were accompanied by downregulation of Kv channel-interacting protein 2 (Kcnip2), a known modulatory subunit for Kv4.2/3 channel. Cardiac VEGF-B overexpression induces a distinct electrophysiological phenotype including remodeling of cardiomyocyte ion currents, which in turn induce changes in action potential waveform and ECG.

M3 cholinoreceptors alter electrical activity of rat left atrium via suppression of L-type Ca2+ current without affecting K+ conductance

Electrophysiological effects produced by selective activation of M3 cholinoreceptors were studied in isolated left atrium preparations from rat using the standard sharp glass microelectrode technique. The stimulation of M3 receptors was obtained by application of muscarinic agonist pilocarpine (10-5 M) in the presence of selective M2 antagonist methoctramine (10-7 M). Stimulation of M3 receptors induced marked reduction of action potential duration by 14.4 ± 2.4% and 16.1 ± 2.5% of control duration measured at 50 and 90% of repolarization, respectively. This effect was completely abolished by selective M3 blocker 4-DAMP (10-8 M). In isolated myocytes obtained from the rat left atrium, similar pharmacological stimulation of M3 receptors led to suppression of peak L-type calcium current by 13.9 ± 2.6% of control amplitude (measured at +10 mV), but failed to affect K+ currents Ito, IKur, and IKir. In the absence of M2 blocker methoctramine, pilocarpine (10-5 M) produced stronger attenuation of ICaL and induced an increase in IKir. This additive inward rectifier current could be abolished by highly selective blocker of Kir3.1/3.4 channels tertiapin-Q (10-6 M) and therefore was identified as IKACh. Thus, in the rat atrial myocardium activation of M3 receptors leads to shortening of action potentials via suppression of ICaL, but does not enhance the major potassium currents involved in repolarization. Joint stimulation of M2 and M3 receptors produces stronger action potential shortening due to M2-mediated activation of IKACh (AU)

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Collagen XIII secures pre- and postsynaptic integrity of the neuromuscular synapse.

Both transmembrane and extracellular cues, one of which is collagen XIII, regulate the formation and function of the neuromuscular synapse, and their absence results in myasthenia. We show that the phenotypical changes in collagen XIII knock-out mice are milder than symptoms in human patients, but the Col13a1-/- mice recapitulate major muscle findings of congenital myasthenic syndrome type 19 and serve as a disease model. In the lack of collagen XIII neuromuscular synapses do not reach full size, alignment, complexity and function resulting in reduced muscle strength. Collagen XIII is particularly important for the preterminal integrity, and when absent, destabilization of the motor nerves results in muscle regeneration and in atrophy especially in the case of slow muscle fibers. Collagen XIII was found to affect synaptic integrity through binding the ColQ tail of acetylcholine esterase. Although collagen XIII is a muscle-bound transmembrane molecule, it also undergoes ectodomain shedding to become a synaptic basal lamina component. We investigated the two forms' roles by novel Col13a1tm/tm mice in which ectodomain shedding is impaired. While postsynaptic maturation, terminal branching and neurotransmission was exaggerated in the Col13a1tm/tm mice, the transmembrane form's presence sufficed to prevent defects in transsynaptic adhesion, Schwann cell invagination/retraction, vesicle accumulation and acetylcholine receptor clustering and acetylcholinesterase dispersion seen in the Col13a1-/- mice, pointing to the transmembrane form as the major conductor of collagen XIII effects. Altogether, collagen XIII secures postsynaptic, synaptic and presynaptic integrity, and it is required for gaining and maintaining normal size, complexity and functional capacity of the neuromuscular synapse.

M3 cholinoreceptors alter electrical activity of rat left atrium via suppression of L-type Ca2+ current without affecting K+ conductance.

Electrophysiological effects produced by selective activation of M3 cholinoreceptors were studied in isolated left atrium preparations from rat using the standard sharp glass microelectrode technique. The stimulation of M3 receptors was obtained by application of muscarinic agonist pilocarpine (10-5 M) in the presence of selective M2 antagonist methoctramine (10-7 M). Stimulation of M3 receptors induced marked reduction of action potential duration by 14.4 ± 2.4% and 16.1 ± 2.5% of control duration measured at 50 and 90% of repolarization, respectively. This effect was completely abolished by selective M3 blocker 4-DAMP (10-8 M). In isolated myocytes obtained from the rat left atrium, similar pharmacological stimulation of M3 receptors led to suppression of peak L-type calcium current by 13.9 ± 2.6% of control amplitude (measured at +10 mV), but failed to affect K+ currents I to, I Kur, and I Kir. In the absence of M2 blocker methoctramine, pilocarpine (10-5 M) produced stronger attenuation of I CaL and induced an increase in I Kir. This additive inward rectifier current could be abolished by highly selective blocker of Kir3.1/3.4 channels tertiapin-Q (10-6 M) and therefore was identified as I KACh. Thus, in the rat atrial myocardium activation of M3 receptors leads to shortening of action potentials via suppression of I CaL, but does not enhance the major potassium currents involved in repolarization. Joint stimulation of M2 and M3 receptors produces stronger action potential shortening due to M2-mediated activation of I KACh.

Peroxisome proliferator-activated receptor-γ coactivator 1 α1 induces a cardiac excitation-contraction coupling phenotype without metabolic remodelling.

KEY POINTS: Transcriptional co-activator PGC-1α1 has been shown to regulate energy metabolism and to mediate metabolic adaptations in pathological and physiological cardiac hypertrophy but other functional implications of PGC-1α1 expression are not known. Transgenic PGC-1α1 overexpression within the physiological range in mouse heart induces purposive changes in contractile properties, electrophysiology and calcium signalling but does not induce substantial metabolic remodelling. The phenotype of the PGC-1α1 transgenic mouse heart recapitulates most of the functional modifications usually associated with the exercise-induced heart phenotype, but does not protect the heart against load-induced pathological hypertrophy. Transcriptional effects of PGC-1α1 show clear dose-dependence with diverse changes in genes in circadian clock, heat shock, excitability, calcium signalling and contraction pathways at low overexpression levels, while metabolic genes are recruited at much higher PGC-1α1 expression levels. These results imply that the physiological role of PGC-1α1 is to promote a beneficial excitation-contraction coupling phenotype in the heart. ABSTRACT: The transcriptional coactivator PGC-1α1 has been identified as a central factor mediating metabolic adaptations of the heart. However, to what extent physiological changes in PGC-1α1 expression levels actually contribute to the functional adaptation of the heart is still mostly unresolved. The aim of this study was to characterize the transcriptional and functional effects of physiologically relevant, moderate PGC-1α1 expression in the heart. In vivo and ex vivo physiological analysis shows that expression of PGC-1α1 within a physiological range in mouse heart does not induce the expected metabolic alterations, but instead induces a unique excitation-contraction (EC) coupling phenotype recapitulating features typically seen in physiological hypertrophy. Transcriptional screening of PGC-1α1 overexpressing mouse heart and myocyte cultures with higher, acute adenovirus-induced PGC-1α1 expression, highlights PGC-1α1 as a transcriptional coactivator with a number of binding partners in various pathways (such as heat shock factors and the circadian clock) through which it acts as a pleiotropic transcriptional regulator in the heart, to both augment and repress the expression of its target genes in a dose-dependent fashion. At low levels of overexpression PGC-1α1 elicits a diverse transcriptional response altering the expression state of circadian clock, heat shock, excitability, calcium signalling and contraction pathways, while metabolic targets of PGC-1α1 are recruited at higher PGC-1α1 expression levels. Together these findings demonstrate that PGC-1α1 elicits a dual effect on cardiac transcription and phenotype. Further, our results imply that the physiological role of PGC-1α1 is to promote a beneficial EC coupling phenotype in the heart.

Age-dependent action of reactive oxygen species on transmitter release in mammalian neuromuscular junctions.

Reactive oxygen species (ROS) are implicated in aging, but the neurobiological mechanisms of ROS action are not fully understood. Using electrophysiological techniques and biochemical assays, we studied the age-dependent effect of hydrogen peroxide (H2O2) on acetylcholine release in rat diaphragm neuromuscular junctions. H2O2 significantly inhibited both spontaneous (measured as frequency of miniature end-plate potentials) and evoked (amplitude of end-plate potentials) transmitter release in adult rats. The inhibitory effect of H2O2 was much stronger in old rats, whereas in newborns tested during the first postnatal week, H2O2 did not affect spontaneous release from nerve endings and potentiated end-plate potentials. Proteinkinase C activation or intracellular Ca2+ elevation restored redox sensitivity of miniature end-plate potentials in newborns. The resistance of neonates to H2O2 inhibition was associated with higher catalase and glutathione peroxidase activities in skeletal muscle. In contrast, the activities of these enzymes were downregulated in old rats. Our data indicate that the vulnerability of transmitter release to oxidative damage strongly correlates with aging and might be used as an early indicator of senescence.

Generation of Functional Neuromuscular Junctions from Human Pluripotent Stem Cell Lines.

Several neuromuscular diseases involve dysfunction of neuromuscular junctions (NMJs), yet there are no patient-specific human models for electrophysiological characterization of NMJ. We seeded cells of neurally-induced embryoid body-like spheres derived from induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) lines as monolayers without basic fibroblast factor (bFGF) and observed differentiation of neuronal as well as spontaneously contracting, multinucleated skeletal myotubes. The myotubes showed striation, immunoreactivity for myosin heavy chain, actin bundles typical for myo-oriented cells, and generated spontaneous and evoked action potentials (APs). The myogenic differentiation was associated with expression of MyoD1, myogenin and type I ryanodine receptor. Neurons formed end plate like structures with strong binding of α-bungarotoxin, a marker of nicotinic acetylcholine receptors highly expressed in the postsynaptic membrane of NMJs, and expressed SMI-32, a motoneuron marker, as well as SV2, a marker for synapses. Pharmacological stimulation of cholinergic receptors resulted in strong depolarization of myotube membrane and raised Ca(2+) concentration in sarcoplasm, while electrical stimulation evoked Ca(2+) transients in myotubes. Stimulation of motoneurons with N-Methyl-D-aspartate resulted in reproducible APs in myotubes and end plates displayed typical mEPPs and tonic activity depolarizing myotubes of about 10 mV. We conclude that simultaneous differentiation of neurons and myotubes from patient-specific iPSCs or ESCs results also in the development of functional NMJs. Our human model of NMJ may serve as an important tool to investigate normal development, mechanisms of diseases and novel drug targets involving NMJ dysfunction and degeneration.

Nrf2 regulates neurogenesis and protects neural progenitor cells against Aß toxicity.

Neural stem/progenitor cells (NPCs) proliferate and produce new neurons in neurogenic areas throughout the lifetime. While these cells represent potential therapeutic treatment of neurodegenerative diseases, regulation of neurogenesis is not completely understood. We show that deficiency of nuclear factor erythroid 2-related factor (Nrf2), a transcription factor induced in response to oxidative stress, prevents the ischemia-induced increase in newborn neurons in the subgranular zone of the dentate gyrus. Consistent with this finding, the growth of NPC neurospheres was increased by lentivirus-mediated overexpression of Nrf2 gene or by treatment with pyrrolidine dithiocarbamate (PDTC), an Nrf2 activating compound. Also, neuronal differentiation of NPCs was increased by Nrf2 overexpression or PDTC treatment but reduced by Nrf2 deficiency. To investigate the impact of Nrf2 on NPCs in Alzheimer's disease (AD), we treated NPCs with amyloid beta (Aß), a toxic peptide associated with neurodegeneration and cognitive abnormalities in AD. We found that Aß1-42-induced toxicity and reduction in neurosphere proliferation were prevented by Nrf2 overexpression, while Nrf2 deficiency enhanced the Aß1-42-induced reduction of neuronal differentiation. On the other hand, Aß1-40 had no effect on neurosphere proliferation in wt NPCs but increased the proliferation of Nrf2 overexpressing neurospheres and reduced it in Nrf2-deficient neurospheres. These results suggest that Nrf2 is essential for neuronal differentiation of NPCs, regulates injury-induced neurogenesis and provides protection against Aß-induced NPC toxicity.

Gender-Specific Mechanism of Synaptic Impairment and Its Prevention by GCSF in a Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motoneurons which progresses differentially in males and females for unknown reason. Here we measured gender differences in pre- and post-synaptic parameters of the neuromuscular transmission in a mutant G93A-SOD1 mouse model of ALS. Using intracellular microelectrode technique we recorded miniature and evoked end-plate potentials (MEPPs and EPPs) in the diaphragm muscle of G93A-SOD1 mice at early symptomatic stage. While no evident alterations in the amplitude of MEPPs was observed in male or female G93A-SOD1 mice, G93A-SOD1 mice displayed dramatically reduced probability of spontaneous acetylcholine release. In contrast, the EPPs evoked by single nerve stimulation had unchanged amplitude and quantal content. In males, but not females, this was accompanied by reduced readily releasable transmitter pool. Transmitter release in both sexes was sensitive to the inhibitory action of reactive oxygen species (ROS), but the production of ROS was increased in the spinal cords of male but not female G93A-SOD1 mice. Treatment with granulocyte colony stimulating factor (GCSF), which we previously found to be beneficial in males, attenuated the increased ROS production indicating involvement of the antioxidant mechanisms and improved ALS-induced synaptic dysfunctions only in males being ineffective in females. Consistent with our findings at synaptic level, GCSF did not change the survival rate or motor performance of female ALS mice. In summary, neuromuscular transmission in ALS mice is impaired at early symptomatic stage when a dramatic presynaptic decline of spontaneous release occurs. Beneficial effects of GCSF treatment on survival in males may be explained by GCSF-improved presynaptic functions in male G93A-SOD1 mice. Development of efficient treatment strategies for ALS may need to be directed in a gender-specific manner.