Nikolay Naumenko

Muscle-Derived Collagen XIII Regulates Maturation of the Skeletal Neuromuscular Junction

Formation, maturation, stabilization, and functional efficacy of the neuromuscular junction (NMJ) are orchestrated by transsynaptic and autocrine signals embedded within the synaptic cleft. Here, we demonstrate that collagen XIII, a nonfibrillar transmembrane collagen, is another such signal. We show that collagen XIII is expressed by muscle and its ectodomain can be proteolytically shed into the extracellular matrix. The collagen XIII protein was found present in the postsynaptic membrane and synaptic basement membrane. To identify a role for collagen XIII at the NMJ, mice were generated lacking this collagen. Morphological and ultrastructural analysis of the NMJ revealed incomplete adhesion of presynaptic and postsynaptic specializations in collagen XIII-deficient mice of both genders. Strikingly, Schwann cells erroneously enwrapped nerve terminals and invaginated into the synaptic cleft, resulting in a decreased contact surface for neurotransmission. Consistent with morphological findings, electrophysiological studies indicated both postsynaptic and presynaptic defects in Col13a1−/− mice, such as decreased amplitude of postsynaptic potentials, diminished probabilities of spontaneous release and reduced readily releasable neurotransmitter pool. To identify the role of collagen XIII at the NMJ, shed ectodomain of collagen XIII was applied to cultured myotubes, and it was found to advance acetylcholine receptor (AChR) cluster maturation. Together with the delay in AChR cluster development observed in collagen XIII-deficient mutants in vivo, these results suggest that collagen XIII plays an autocrine role in postsynaptic maturation of the NMJ. Altogether, the results presented here reveal that collagen XIII is a novel muscle-derived cue necessary for the maturation and function of the vertebrate NMJ.

Cortical spreading depression induces oxidative stress in the trigeminal nociceptive system.

Indirect evidence suggests the increased production of reactive oxygen species (ROS) in migraine pathophysiology. In the current study we measured lipid peroxidation product in the rat cortex, trigeminal ganglia and meninges after the induction of cortical spreading depression (CSD), a phenomenon known to be associated with migraine aura, and tested nociceptive firing triggered by ROS in trigeminal nerves ex vivo. Application of KCl to dura mater in anesthetized rats induced several waves of CSD recorded by an extracellular electrode in the cortex. Following CSD, samples of cortex (affected regions were identified with blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI)), meninges from left and right hemispheres and trigeminal ganglia were taken for biochemical analysis. We found that CSD increased the level of the lipid peroxidation product malondialdehyde (MDA) in the ipsilateral cerebral cortex and meninges, but also in both ipsi- and contralateral trigeminal ganglia. In order to test the pro-nociceptive action of ROS, we applied the mild oxidant hydrogen peroxide to isolated rat hemiskull preparations including preserved trigeminal innervations. Application of hydrogen peroxide to meninges transiently enhanced electrical spiking activity of trigeminal nerves showing a pro-nociceptive action of ROS. In the presence of hydrogen peroxide trigeminal nerves still responded to capsaicin by burst of spiking activity indicating integrity of neuronal structures. The action of hydrogen peroxide was mediated by TRPA1 receptors as it was abolished by the specific TRPA1 antagonist TCS-5861528. Using dorsal root ganglion sensory neurons as test system we found that hydrogen peroxide promoted the release of the migraine mediator calcitonin gene-related peptide (CGRP), which we previously identified as a trigger of delayed sensitization of trigeminal neurons. Our data suggest that, after CSD, oxidative stress spreads downstream within the trigeminal nociceptive system and could be involved in the coupling of CSD with the activation of trigeminovascular system in migraine pathology.

Nrf2 Regulates Neurogenesis and Protects Neural Progenitor Cells Against Aβ 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 Alzheimers disease (AD), we treated NPCs with amyloid beta (Aβ), a toxic peptide associated with neurodegeneration and cognitive abnormalities in AD. We found that Aβ1–42‐induced toxicity and reduction in neurosphere proliferation were prevented by Nrf2 overexpression, while Nrf2 deficiency enhanced the Aβ1–42‐induced reduction of neuronal differentiation. On the other hand, Aβ1–40 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 Aβ‐induced NPC toxicity. Stem Cells 2014;32:1904–1916

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.

SNARE tagging allows stepwise assembly of a multimodular medicinal toxin

Generation of supramolecular architectures through controlled linking of suitable building blocks can offer new perspectives to medicine and applied technologies. Current linking strategies often rely on chemical methods that have limitations and cannot take full advantage of the recombinant technologies. Here we used SNARE proteins, namely, syntaxin, SNAP25, and synaptobrevin, which form stable tetrahelical complexes that drive fusion of intracellular membranes, as versatile tags for irreversible linking of recombinant and synthetic functional units. We show that SNARE tagging allows stepwise production of a functional modular medicinal toxin, namely, botulinum neurotoxin type A, commonly known as BOTOX. This toxin consists of three structurally independent units: Receptor-binding domain (Rbd), Translocation domain (Td), and the Light chain (Lc), the last being a proteolytic enzyme. Fusing the receptor-binding domain with synaptobrevin SNARE motif allowed delivery of the active part of botulinum neurotoxin (Lc-Td), tagged with SNAP25, into neurons. Our data show that SNARE-tagged toxin was able to cleave its intraneuronal molecular target and to inhibit release of neurotransmitters. The reassembled toxin provides a safer alternative to existing botulinum neurotoxin and may offer wider use of this popular research and medical tool. Finally, SNARE tagging allowed the Rbd portion of the toxin to be used to deliver quantum dots and other fluorescent markers into neurons, showing versatility of this unique tagging and self-assembly technique. Together, these results demonstrate that the SNARE tetrahelical coiled-coil allows controlled linking of various building blocks into multifunctional assemblies.

Hunting for origins of migraine pain: cluster analysis of spontaneous and capsaicin-induced firing in meningeal trigeminal nerve fibers.

Trigeminal nerves in meninges are implicated in generation of nociceptive firing underlying migraine pain. However, the neurochemical mechanisms of nociceptive firing in meningeal trigeminal nerves are little understood. In this study, using suction electrode recordings from peripheral branches of the trigeminal nerve in isolated rat meninges, we analyzed spontaneous and capsaicin-induced orthodromic spiking activity. In control, biphasic single spikes with variable amplitude and shapes were observed. Application of the transient receptor potential vanilloid 1 (TRPV1) agonist capsaicin to meninges dramatically increased firing whereas the amplitudes and shapes of spikes remained essentially unchanged. This effect was antagonized by the specific TRPV1 antagonist capsazepine. Using the clustering approach, several groups of uniform spikes (clusters) were identified. The clustering approach combined with capsaicin application allowed us to detect and to distinguish “responder” (65%) from “non-responder” clusters (35%). Notably, responders fired spikes at frequencies exceeding 10 Hz, high enough to provide postsynaptic temporal summation of excitation at brainstem and spinal cord level. Almost all spikes were suppressed by tetrodotoxin (TTX) suggesting an involvement of the TTX-sensitive sodium channels in nociceptive signaling at the peripheral branches of trigeminal neurons. Our analysis also identified transient (desensitizing) and long-lasting (slowly desensitizing) responses to the continuous application of capsaicin. Thus, the persistent activation of nociceptors in capsaicin-sensitive nerve fibers shown here may be involved in trigeminal pain signaling and plasticity along with the release of migraine-related neuropeptides from TRPV1 positive neurons. Furthermore, cluster analysis could be widely used to characterize the temporal and neurochemical profiles of other pain transducers likely implicated in migraine.

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

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.

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

Summary Alzheimers 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 β-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.

PGC-1α1 induces cardiac excitation-contraction coupling phenotype without metabolic remodelling.

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.