Prenatal hyperhomocysteinemia induces oxidative stress and accelerates 'aging' of mammalian neuromuscular synapses

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(1)UEF//eRepository DSpace Rinnakkaistallenteet. https://erepo.uef.fi Terveystieteiden tiedekunta. 2019. Prenatal hyperhomocysteinemia induces oxidative stress and accelerates 'aging' of mammalian neuromuscular synapses Khuzakhmetova, V Elsevier BV Tieteelliset aikakauslehtiartikkelit © ISDN CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ http://dx.doi.org/10.1016/j.ijdevneu.2019.03.004 https://erepo.uef.fi/handle/123456789/7584 Downloaded from University of Eastern Finland's eRepository.

(2) Accepted Manuscript Title: Prenatal hyperhomocysteinemia induces oxidative stress and accelerates ‘aging’ of mammalian neuromuscular synapses Authors: Venera Khuzakhmetova, Olga Yakovleva, Svetlana Dmitrieva, Nail Khaertdinov, Guzel Ziyatdinova, Rashid Giniatullin, Aleksey Yakovlev, Ellya Bukharaeva, Guzel Sitdikova PII: DOI: Reference:. S0736-5748(18)30302-2 https://doi.org/10.1016/j.ijdevneu.2019.03.004 DN 2342. To appear in:. Int. J. Devl Neuroscience. Received date: Revised date: Accepted date:. 19 October 2018 6 February 2019 21 March 2019. Please cite this article as: Khuzakhmetova V, Yakovleva O, Dmitrieva S, Khaertdinov N, Ziyatdinova G, Giniatullin R, Yakovlev A, Bukharaeva E, Sitdikova G, Prenatal hyperhomocysteinemia induces oxidative stress and accelerates ‘aging’ of mammalian neuromuscular synapses, International Journal of Developmental Neuroscience (2019), https://doi.org/10.1016/j.ijdevneu.2019.03.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain..

(3) Prenatal hyperhomocysteinemia induces oxidative stress and accelerates ‘aging’ of mammalian neuromuscular synapses. Venera Khuzakhmetova1,2, Olga Yakovleva2, Svetlana Dmitrieva1, Nail Khaertdinov2, Guzel Ziyatdinova2, Rashid Giniatullin2,3, Aleksey Yakovlev2, Ellya Bukharaeva1,2, Guzel Sitdikova 2* 1. IP T. Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, Kazan, Russia. 2 Kazan Federal University, Kazan, Russia 3 A.I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland *Corresponding. SC R. author: Guzel Sitdikova Department of Human and Animal Physiology, Institute of Fundamental Biology and Medicine, Kazan Federal University, Kazan, 420008, Kremlevskaya str., 18, Russia Email: sitdikovaguzel@gmail.com. Highlights.  . A. M.  . High intensity of quantal release was shown in synapses of neonatal rats with prenatal hHCY Prenatal hHCY induces synchronization of quantal release in synapses of newborn rats Synapses of neonates with hHCY are susceptible to inhibitory effects of hydrogen peroxide Prenatal hHCY is resulted in the appearance of adult-like synapses in newborn rats Prenatal hHCY induces malfunctioning of the antioxidant defense in rat diaphragm muscle. ED. . N. U. 3 to 5 bullet points (maximum 85 characters, including spaces, per bullet point).. A. CC E. PT. Enhanced levels of homocysteine during pregnancy induce oxidative stress and contribute to many age-related diseases. In this study, we analyzed age-dependent synaptic modifications in developing neuromuscular synapses of rats with prenatal hyperhomocysteinemia (hHCY). One of the main findings indicate that the intensity and the timing of transmitter release in synapses of neonatal (P6 and P10) hHCY rats acquired features of matured synaptic transmission of adult rats. The amplitude and frequency of miniature end-plate currents (MEPCs) and evoked transmitter release were higher in neonatal hHCY animals compared to the control group. Analysis of the kinetics of neurotransmitter release demonstrated more synchronized release in neonatal rats with hHCY. At the same time lower release probability was observed in adults with hHCY. Spontaneous transmitter release in neonates with hHCY was inhibited by hydrogen peroxide (H2O2) whereas in controls this oxidant was effective only in adult animals indicating a higher susceptibility of motor nerve terminals to oxidative stress. The morphology and the intensity of endocytosis of synaptic vesicles in motor nerve endings was assessed using the fluorescence dye FM1-43. Adult-like synapses were found in neonates with hHCY which were characterized by a larger area of presynaptic terminals compared to controls. No difference in the intensity of FM1-43 fluorescence was observed between two groups of animals. Prenatal hHCY resulted in reduced muscle strength assessed by the Paw Grip Endurance test. Using biochemical assays we found an increased level of H2O2 and lipid peroxidation products in the diaphragm 1.

(4) muscles of hHCY rats. This was associated with a lowered activity of superoxide dismutase and glutathione peroxidase. Our data indicate that prenatal hHCY induces oxidative stress and apparent faster functional and morphological "maturation" of motor synapses. Our results uncover synaptic mechanisms of disrupted muscle function observed in hHCY conditions which may contribute to the pathogenesis of motor neuronal diseases associated with enhanced level of homocysteine. Abbreviations: hHCY, hyperhomocysteinemia; SOD, superoxide dismutase; GPx, glutathione peroxidase; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; HCY, homocysteine; ALS, amyotrophic lateral sclerosis; ROS, reactive oxygen species; MDA, malondialdehyde; MEPCs, miniature end-plate currents; EPCs, end-plate currents; PaGE, Paw Grip Endurance. SC R. IP T. Keywords Prenatal hyperhomocysteinemia, neonatal and adult rats, developing neuromuscular junction, spontaneous and evoked release, kinetic of quantum release, oxidative stress, muscle strength 1. INTRODUCTION. A. CC E. PT. ED. M. A. N. U. Homocysteine (НСY) is a sulfur containing amino acid synthesized from methionine (Petras et al., 2014; Sharma et al., 2015; Veeranki and Tyagi, 2013). Impairments of the HCY metabolism induce increased HCY plasma levels called hyperhomocysteinemia (hHCY). The normal range of HCY in the blood plasma is 5 - 12 µM. Elevation of HCY level up to 15 - 25 µM induces mild hHCY. 25–50 µM of HCY corresponds to moderate hHCY and higher concentrations (>100 µM) are considered as severe form of hHCY in case of inborn failures of homocysteine metabolism (Jacobsen, 1998; Sharma et al., 2015). hHCY is often followed by skeletal muscle dysfunctions, evidenced by muscle weakness, less fatigue resistance due to oxidative stress, inflammation and endoplasmic reticulum stress in mice (Swart et al., 2013; Schweinberger et al., 2014; Veeranki and Tyagi, 2015; Majumder et al., 2017; 2018a,b). The elevated levels of HCY are associated with neurodegenerative disorders and amyotrophic lateral sclerosis (ALS) (McCully, 2017; Sharma et al., 2015; Swart et al., 2013). Oxidative stress is one of the mechanisms of the damaging action of HCY known as a powerful oxidant, producing reactive oxygen species (ROS) such as superoxide anion (O2-) and hydrogen peroxide (H2O2) (Petras, 2014), which affect synaptic transmission at central and peripheral nervous systems (Giniatullin et al., 2006). Recently it was shown that HCY sensitizes neuromuscular junction to the inhibitory effect of H2O2 via activation of NMDA receptors and nitric oxide (Bukharaeva et al., 2015; Wang et al., 2018). Elevated levels of HCY during pregnancy (prenatal hHCY) impair fetus development, resulted in functional disabilities, learning deficits and skeletal muscle myopathy of the offspring (Hague, 2003; Picker and Levy, 2010; Gerasimova et al., 2017; Makhro et al., 2008; Yakovlev et al., 2018; Yakovelva et al., 2018). Newborns are particularly vulnerable to oxidative stress due to high ability of ROS production and decreased antioxidant protection (Marseglia et al., 2014). Although mechanisms of detrimental effects of HCY are intensively studied in skeletal muscles, the function of peripheral synapses during hHCY conditions has not been investigated. The aim of the present study was to analyze the processes of synaptic transmission in neonatal and adult rats with maternal hHCY. Namely, we studied: (1) the intensity and kinetics of spontaneous and evoked quantal transmitter release from motor nerve endings; (2) the effects of H2O2 on synaptic transmission; (3) the shape and area of presynaptic terminals and the intensity of endocytosis of synaptic vesicles using fluorescence microscopy; (4) muscle strength measured with a the Paw Grip Endurance (PaGE) test, and (5) ROS production and the activity of the antioxidant enzymes in diaphragm muscles. 2. MATERIAL AND METHODS 2.

(5) 2.1 Experimental animals and the model of hHCY. IP T. All animal experiments were performed in accordance with the European Community Council Directive of September 22, 2010 (2010/63/EEC) and approved by the Ethics Committee of Kazan Federal University. Animals were anesthetized with isofluran before decapitation. Experiments were performed on isolated phrenic nerve-diaphragm muscle preparations from Wistar rats of both sexes of 6 days (P6), P10 after birth and at the age of 10 - 12 weeks (adults). Pups for control experiments were born from females fed ad libitum with a control diet. Pups with prenatal hHCY were born from females which received daily methionine (7.7 g/kg body weight) with food starting three weeks prior to and during pregnancy (Gerasimova et al., 2017). The total HCY level in plasma was detected by voltammetry measurements of products of its reactions with о-quinone (Lee et al., 2014; Gerasimova et al., 2017). SC R. 2.2 Electrophysiology and data analysis. A. CC E. PT. ED. M. A. N. U. An isolated phrenic nerve–diaphragm muscle preparation was pinned to the bottom of a 5 ml chamber and superfused with Krebs–Ringer solution of the following composition (mM): 120.0 NaCl, 5.0 KCl, 0.4 CaCl2, 11.0 NaHCO3, 1.0 NaHPO4, 5.0 MgCl2, 11 glucose, pH 7.2 7.4. The solution was bubbled with 95% O2 and 5% CO2. The experiments were performed at 20.0 ± 0.50C. Low Ca2+ and high Mg2+ containing solution was used to avoid the muscle contractions in response to motor nerve stimulation and in order to measure synaptic delays of uni-quantal endplate currents (EPCs). Presynaptic action potentials and EPCs were recorded using heat-polished Ringer filled extracellular pipettes with tip diameters of 2 - 3 µM and resistances of 1 - 3 MΩ which were positioned at the nerve ending under visual control (Olympus BW-51, Japan) (Brigant and Mallart, 1982; Gerasimova et al., 2015). Stimulation of the motor nerve was performed by suprathreshold stimuli with durations of 0.1 ms at 0.5 Hz via a suction electrode. The recorded signals were filtered between 0.03 Hz and 10 kHz, digitized at 3-5 µs intervals with a 16-bit analog–digital converter, sampled, stored in a computer and processed using our application package (Bukcharaeva et al., 1999). L-HCY and hydrogen peroxide 50% were obtained from Sigma (St. Louis, MO, USA). The amplitude, rise time (RT), decay time (τ) of 100 - 150 miniature end-plate currents (MEPCs) were sampled to assess spontaneous quantal release before nerve stimulation. Analysis of parameters of the evoked responses was performed in the time «window» of 50 ms. Probability of MEPCs was calculated in each experiment. In case the probability was higher than 0.05 (5 signals for 100 stimuli), the experiment was not used for analysis. The average quantal content of EPCs (m) was calculated using the “failures” method (Martin, 1955) according to the equation m=ln N/nо, where N – is the number of stimuli and nо the number of failures. Synaptic delays were measured as the time interval between the peak of the inward presynaptic current and the point at which the rise time of EPCs reached 20% of their maximum (Katz and Miledi, 1965; Khuzakhmetova et al., 2014a). For analysis of synaptic delays at least 300 uni-quantal EPCs were recorded. The minimal synaptic delay was determined as the mean values of the shortest 5% delays in each series (Bukcharaeva et al., 1999; Khuzakhmetova et al., 2014a). For analysis of synchronous and asynchronous release the number of quanta released during first 3 ms (the early phase of synchronous release), next 5 ms (the late phase of synchronous release) and in the time interval from 8 to 50 ms (delayed asynchronous phase of quantal release) were assessed (Chen and Regehr, 1999; Khuzakhmetova et al., 2014a; Vasin et al., 2010). 2.3Fluorescent microscopy. 3.

(6) A. N. U. SC R. IP T. The neuromuscular preparation was placed into a bath perfused by the oxygenated Krebs solution containing (in mM): 137.0 NaCl; 5.0 KCl; 2.2 CaCl2; 1.0 MgCl2; 1.0 NaH2PO4; 16.0 NaHCO3; 11.0 glucose; oxygenated with 95% O2 and 5% CO2 (at 20°C, pH 7.2 - 7.4). For analysis of nerve endings morphology and intensity of endocytosis of synaptic vesicles the fluorescent styryl dye FM 1-43 (Biotium, USA, 3 µm) was used. FM 1-43 reversibly binds to the presynaptic membrane and becomes trapped within recycled synaptic vesicles during endocytosis. High frequency stimulation of the motor nerve (50 Hz for 1 min) was used to load the nerve terminals with the dye. FM 1-43 was present in the solution for 1 min during and 7 min after stimulation followed by washout of dye with Krebs solution containing ADVASEP-7 (Biotium, USA, 3 μm) to decrease nonspecific fluorescence (Petrov et al., 2010; Yakovleva et al., 2017). The fluorescence of nerve terminals reflects the intensity of synaptic vesicle endocytosis. The terminals’ staining was calculated in arbitrary units (a.u.) after subtraction of background fluorescence. The background fluorescence value was defined as an average brightness of the image section outside the terminals. FM 1-43 staining also allowed us to assess the shape and area of nerve terminals (Petrov et al., 2010). Observations were conducted only on the surface nerve endings. Control and hHCY rats of different age groups (P6, P10 and P18) were used. The age P18 was chosen as at this age the diaphragm muscles are still thin and transparent enough to observe the surface nerve terminals using fluorescence microscopy. Moreover, at this age the adult-like synapses are fully developed which subsequently only increase in size without changes of their shape (Sanes and Lichtman, 1999) and the neurotransmitter release pattern corresponds to older animals (Khuzakhmetova et al., 2014a). Fluorescence staining of nerve endings was recorded using an AxioScope A1 microscope equipped with a high-speed monochrome camera AxioCam MRm (Carl Zeiss, Germany). Image analysis was performed using programs Image J software (National Institutes of Health, Bethesda, MD, USA) and Image Pro Plus (Media Cybernetics, USA).. M. 2.4 Muscle strength. PT. ED. Muscle strength was assessed by the Paw Grip Endurance (PaGE) Test (Weydt et al., 2003; Yakovleva et al., 2018) at P10 and adults. Rats were placed on a wire grid and gently shaken to prompt the rat to grip the grid. The lid was turned upside down over a housing cage and held at ~0.45 m above an open cage bottom. The time (s) spent on the grid before falling was assessed. The largest value from three individual trials was used for analysis. 2.5 Detection of an oxidative stress level. CC E. 2.5.1 Measurement of hydroperoxides and malondialdehyde. A. Concentrations of Н2О2 were measured by ferrous oxidation in xylenol orange (FOX1 reagent) using spectrophotometer Lambda-25 (Perkin Elmer, USA) as described by Wolff (Wolff, 1994). FOX1 reagent contained (in mM): 0.5 ZnSO4, 0.5 (NH4)2SO4, 50 H2SO4, 0.2 xylenol orange, 200 D-sorbitol (Sigma, USA). Samples of diaphragm muscle were fixed in liquid nitrogen, then homogenized in cold buffer solution (0.1 M 4-morpholineethanesulfonic acid at pH 6.0, at a ratio of 1:10) and centrifuged for 10 minutes at 10,000 g. The supernatant was mixed with FOX1 reagent at equal volumes. After 30 min incubation the supernatant was assayed spectrophotometrically (λ = 560 nm). Each measurement was made at least 3 times and then averaged. The peroxide content in samples was determined with reference to a calibration curve obtained with known concentrations of H2O2. The level of H2O2 was expressed as µg/g of tissues. Malondialdehyde (MDA), a marker of lipid peroxidation, was determined spectrophotometrically according to the method of Ohkawa et al. (1979). At low pH and elevated temperature, MDA readily participates in nucleophilic addition reaction with 2-thiobarbituric 4.

(7) acid (TBA), generating a red, fluorescent 1:2 MDA adduct. After homogenization, the tissue homogenate was mixed at a ratio of 1:1 with 0.3 % Triton Х-100, 0.1 М НСl, 0.03 М ТBA. The mixture was heated for 45 min at 95°C and centrifuged for 10 minutes at 10,000 g. The absorbance of the supernatant was monitored at 532 nm and at 560 nm (εTBA-MDA =1.55 mM-1 cm-1). MDA levels were expressed as µg/g of tissues. 2.5.2 Antioxidant enzyme activity. CC E. PT. ED. M. A. N. U. SC R. IP T. Antioxidant potential was determined by measuring activities of catalase, glutathione peroxidase (GPx), superoxide dismutase (SOD) in cytosolic fractions of diaphragm muscle preparations. The samples of diaphragm muscle were fixed in liquid nitrogen, then homogenized in cold buffer solution (0.1 M MES at pH 6.0, at a ratio of 1:10) and centrifuged for 10 minutes at 10,000 g. Catalase activity was assayed by measuring the decrease in absorbance due to H2O2 consumption (εH2O2 = 43.6 M-1 cm-1) at 240 nm (Fulle et al., 2005). The reaction mixture contained 50 mM Na2HPO4 buffer (pH 7.0), 40 mM H2O2 and 150 ml sample. The reaction was initiated by the addition of H2O2. Enzyme activity is reported as units per min per mg of protein (UCAT/min/mg). SOD activity (Cu/Zn superoxide dismutase) was determined according to Weydert and Cullen (2010). In this method a xanthine/xanthine oxidase system is used to generate O2− and nitroblue tetrazolium (NBT) reduction serves as an indicator of O2− production. SOD competes with NBT for O2−. The percent inhibition of NBT reduction reflects the amount of SOD, which is assayed in a spectrophotometer at 560 nm. The reaction mixture contained 100 mM Na2HPO4 buffer (pH 10.2), 0.1 mM EDTA, 1 M cytochrome c, 1 mM xanthine, 0.04 mM NBT and 150 µl sample. The reaction was initiated by the addition of 0.05 unit xanthine oxidase. The inhibition of the produced chromogen is proportional to the activity of SOD present in the sample. A 50% inhibition is defined as 1 unit of SOD, and specific activity is expressed as units per min per mg of protein (USOD/min/mg). GPx activity was determined according to Weydert and Cullen (2010). GPx catalyzes the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione reductase and NADPH, the oxidized glutathione (GSSG) is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+. The decrease in absorbance was monitored with a spectrophotometer at 340 nm. The reaction mixture consisted of 50 mM Na2HPO4 buffer (pH 7.2), 1 mM reduced glutathione (GSH), 0.5 units of glutathione reductase, 0.15 mM NADPH, 1mM EDTA and 150 µl sample. One GPx unit is defined as 1 µmol of GSH consumed per minute, and activity is reported as units per min per mg of protein (UPOX/min/mg). Protein content was measured using Bradfords assay (Bradford, 1976) using bovine serum albumin as standard. A volume of 20 µl of sample or standard was mixed with 1 ml Bradford reagent, and the absorbance was assessed by a spectrophotometer at 595 nm after 5 min.. A. 2.6 Statistical analysis Normality of sample data was evaluated with the Shapiro-Wilk test and for equal variances Ftest Origin Pro software (OriginLab Corp, USA) was used. Statistical significance was assessed by using Student’s t-test (for parametric data) and Mann-Whitney test (for nonparametric data). Differences were considered as statistically significant at p < 0.05 in at least four independent experiments. Data are presented as mean ± SEM, n – indicates the number of synapses and N – number of animals. 3 RESULTS 5.

(8) 3.1 Plasma HCY concentrations Concentration of HCY in the plasma of control females was 7.0 ± 1.0 µM (N = 14) and in females fed with methionine contained diet 30.0 ± 5.0 µM (N = 14). Concentration of HCY in adult rats born from females fed with methionine contained diet was 21.7 ± 2.8 µM (N = 16) and born from control animals - 6.23 ± 0.42 µM (N = 9). 3.2 Spontaneous quantal release in hHCY. PT. ED. M. A. N. U. SC R. IP T. First, in control and hHCY rats, we tested amplitudes and frequency of MEPCs which reflect the sensitivity of the postsynaptic membrane and the intensity of spontaneous quantal release, respectively. We found that the amplitude of spontaneous MEPCs in newborns from the hHCY group was about 50% higher compared to the control group but no differences were observed in P10 and adult animals (Fig. 1 A, B; Table 1). The temporal parameters of MEPCs such as the rise time (RT) and the decay time constant (τ) progressively decreased in both groups during postnatal development. However, in the synapses of P6 rats with hHCY the RT and τ were much shorter compared to the control group indicating a shorter open-state lifetime of the postsynaptic acetylcholine receptor channel (Table 1; Katz and Miledi, 1973). In both groups, MEPC frequency was significantly lower in newborn animals compared to adults (Fig. 1 C). However, in animals with prenatal hHCY, at P10 and adults, the frequency of MEPCs was higher compared to control rats of the same age (Fig. 1 C).. A. CC E. Fig. 1. Spontaneous quantal release at neuromuscular junctions of control and hHCY rats. (A) Average traces of MEPCs in control and hHCY of Р6, Р10 and adult animals. Mean amplitudes (B) and frequencies (C) of MEPCs in control (white columns) and hHCY animals (grey columns). In control: P6 – n = 11, N = 10; P10 – n = 16, N = 14; adults – n = 11, N = 8. In hHCY: P6 – n = 12, N = 7; P10 – n = 14, N = 7; adults – n = 9, N = 5. *p < 0.05 compared to control.. Contr ol. Table 1. Amplitudes and temporal parameters of MEPCs in synapses of Р6, Р10 and adult rats from control and hHCY groups. MEPCs Amplitude Rise time Tau (mV) (ms) (ms) P6. 0.10±0.02. 0.41±0.22. 5.20±0.56 6.

(9) 0.12±0.02. 0.36±0.12. 2.82±0.22. Adult. 0.13±0.01. 0.27±0.01. 1.59±0.08. P6. 0.15±0.02*. 0.35±0.01*. 3.08±0.20*. P10. 0.18±0.03. 0.35±0.02. 2.35±0.15. Adult. 0.12±0.01. 0.27±0.01. 1.35±0.09. IP T. hHCY. P10. The number of synapses/animals is the same as at the Fig. 1. * p < 0.05 compared to control. SC R. 3.3 Evoked transmitter release in hHCY. A. CC E. PT. ED. M. A. N. U. The probability of evoked release was evaluated by the analysis of the amplitudes, RT, τ and quantal content of EPCs elicited by low frequency nerve stimulation. We found that at the synapses of P6 and P10 animals with prenatal hHCY amplitudes and quantal content of EPCs were significantly higher compared to control groups (Fig. 2 A; Table 2). At P6 with hHCY the quantal content of EPCs was 2 times higher compared to control (0.58 ± 0.13, n = 9, N = 5 vs 0.29 ± 0.03, n = 30, N = 26, p < 0.05; Fig. 2 A). At P10, the quantal content of EPCs was 0.22 ± 0.02 (n = 26, N = 21) in control and 0.34 ± 0.06 in hHCY animals (n = 12, N = 7, p < 0.05). However, in synapses of adult hHCY animals the quantal content of EPCs was lower than in control (0.47 ± 0.06, n = 11, N = 5 vs 0.71 ± 0.09, n = 17, N = 14, p < 0.05; Fig. 2 A). Temporal parameters of EPCs (RT and τ) at P6 animals were shorter compared to the control group in agreement with similar changes of MEPCs indicating of the faster “maturation” of synapses in hHCY conditions (Table 2).. Fig. 2. Evoked transmitter release in control and hHCY animals of neonatal and adult groups. The average quantal content of EPCs (A) and average minimal synaptic delay of EPCs (B) control (white columns) and hHCY (grey columns) in synapses from newborn and adult animals. In control: P6 – n = 30, N = 26; P10 – n = 26, N = 21; adults – n = 17, N = 14. In hHCY: P6 – n = 9, N = 5; P10 – n = 12, N = 7; adults – n = 11, N = 6. * p < 0.05 compared to control. 7.

(10) IP T. SC R. Table 2. Amplitudes and temporal parameters of EPCs in synapses of Р6, Р10 and adult rats from control and hHCY groups.. Rise time (ms). Tau (ms). P6. 0.10±0.01. 0.35±0.15. 3.36± 0.29. P10. 0.13±0.01. Adult. 0.12±0.01. P6. 0.17±0.02*. Adult. PT. hHCY. P10. N. U. Amplitude (mV). A. 0.32±0.09. 2.27±0.13 1.53±0.07. 0.30±0.01*. 2.33±0.15*. 0.16±0.01*. 0.29±0.01. 2.11±0.12. 0.12±0.01. 0.27±0.1*. 1.34±0.06*. M. 0.31±0.01. ED. Control. EPCs. CC E. The number of synapses/animals is the same as at the Fig. 2. * p < 0.05 compared to control 3.4 The timing of synaptic events in hHCY. A. Low concentrations of extracellular calcium allowed us not only to estimate the probability of the quantal release, but also assess the timing of transmitter release (Barrett and Stevens, 1972; Bukcharaeva et al., 1999; Katz and Miledi, 1965). We found that in the hHCY group at Р6 minimal synaptic delays were shorter compared to control animals of the same age (Fig. 2 B). Synaptic delays of uni-quantal responses in our conditions essentially varied, reflecting an asynchronous release of individual quanta which was more visible in newborns (Fig. 3; Khuzakhmetova et al., 2014a, b). In control synapses of P6 animals most quanta were released during late phasic and delayed asynchronous periods indicated asynchronous release (Fig 3A, Table 3). With growing most quanta released during phasic period indicated a progressive synchronization of the secretory process (Fig. 3 C-F, Table 3). However, in synapses of P6 and P10 animals from the hHCY group, the number of quanta released during early/phasic 8.

(11) A. CC E. PT. ED. M. A. N. U. SC R. IP T. release was 2.5 and 1.6 times larger compared to controls, respectively (Fig. 3 A-D, Table 3). In adult hHCY animals quantal release during the early synchronous period was lower compared to the control group which is in accordance with the lower quantal content of EPCs (Fig 3E, F, Table 3). These results indicate a higher level of synchronization of quantal release in neonatal hHCY animals which is a typical characteristic of the mature synapses. Acute application of 500 µM HCY at neuromuscular preparations of control animals for 30 min did not change the temporal parameters and intensity of spontaneous and evoked acetylcholine release in synapses of newborn (P6 and P10) and adult animals.. Fig. 3. Synchronization of the secretory process in rats from control and hHCY groups. Histograms of synaptic delays of uni-quantal EPCs in synapses of P6 (A), P10 (C) and adult (E) animals. Black columns – control group, grey columns – hHCY group. Bin size is 0.5 ms. 9.

(12) Inserts: superposition of extracellularly recorded uni-quantal EPCs (8 - 10 traces) recorded at P6, P10 and mature synapses at 0.5 Hz stimulation frequency from animals of control (black line) and hHCY (grey line) groups. The traces with failures are not shown. Scale bars - 0.1 mV and 5 ms. Cumulative plots of synaptic delays of uni-quantal EPCs from synapses of P6 (B), P10 (D) and adult (F) animals. Inserts: cumulative plots of synaptic delays during first 10 ms of release period. ER - early synchronous release (0-3 ms); LR - late synchronous release (3-8 ms); DR delayed asynchronous release (8-50 ms).. IP T. Table 3. The number of quanta released during different periods of secretion in response to 1000 stimuli in synapses of Р6, Р10 and adult rats from control and hHCY groups. Early synchronous release (0 – 3 ms). Late synchronous release (3 - 8 ms). Delayed asynchronous release (8 - 50 ms). Control. hHCY. Control. hHCY. Control. Р6. 79±12. 198±38*. 209±30. 386±103* 105±16. 190±50*. Р10. 105±14. 172±20*. 125±14. 132±43. 71±9. 111±36. Adult. 667±83. 412±69*. 41±5. 155±27. 203±28. SC R. U. N. A. 51±7. hHCY. M. In control: P6 – n = 30, N = 26; P10 – n = 26, N = 21; adults – n = 17, N = 14. In hHCY: P6 – n = 9, N = 5; P10 – n = 12, N = 7; adults – n = 9, N = 5. * p < 0.05 compared to control.. ED. 3.5 The effects of H2O2 on synaptic transmission of rats from hHCY and control groups. A. CC E. PT. As was shown recently the stable and membrane permeable oxidant H2O2, agedependently affects both spontaneous and evoked transmitter release in rat neuromuscular junctions (Shakirzyanova et al., 2016). In our experiments 300 µМ H2O2 reduced MEPCs frequency only in adult rats (64 ± 9 % of control, Fig. 4, n = 7, N = 5). In contrast, in the hHCY group H2O2 decreased the intensity of spontaneous release in synapses of animals of all tested ages. At P6 the frequency of MEPCs was reduced to 53 ± 10 % (n = 5, N = 4; p < 0.05), at P10 to 46 ± 7 % (n =7, N = 6; p < 0.05) and at adult animals - to 15 ± 4 % (n = 5, N = 4; p < 0.05) compared to controls, respectively (Fig. 4). H2O2 had no significant effects on the probability and timing of quantal events in both groups of animals (Table S1). There was only a decrease in delayed asynchronous release in Р6 and Р10 animals from the hHCY group. In the control group H2O2 did not change the timing of evoked secretion (Table S1).. 10.

(13) IP T SC R. N. U. Fig. 4. H2O2 action on spontaneous quantal release in synapses of control and hHCY animals. Changes of MEPCs frequency after H2O2 (300 µM) application at neuromuscular junctions of neonatal and adult rats (% of initial level) of control (white columns) and hHCY (grey columns) groups. In control: P6 – n = 7, N = 6; P10 – n = 5, N = 4; adults – n = 7, N = 5. In hHCY: P6 – n = 5, N = 4; P10 – n = 7, N = 6; adults – n = 5, N = 4. * p < 0.05 – compared to values before application of H2O2.. M. A. 3.6 Morphology of nerve terminals and the intensity of endocytosis in synapses of rats of control and hHCY groups. A. CC E. PT. ED. In our experiments we observed different types of presynaptic terminals staining with FM 1-43 (Fig. 5A). At P6 plaque-like synapses with uniform fluorescence (type 1) were visualized. At P10 few branches and perforations of nerve terminals with discrete bright active zones (type 2) appeared. At P18 branched terminal arbors with multiple active zones (type 3) reflecting the maturation of synaptic contacts to adult forms were observed (Fig. 5 A). We assessed the ratio of three types of nerve terminals in different ages (Fig. 5 B). At P6 about 50% of all observed terminals were immature (type 1). At the same time in P6 hHCY rats 40% of all terminals developed to the adult forms (type 3). The increased number of adult-like synapses was also observed at P10 hHCY animals (Fig. 5 B). At the same time at P18 the fraction of adult-like synapses was larger in control group (type 3), indicating on the delay of synapse development during third postnatal week (Fig. 5 B). The average area of presynaptic terminals in control rats increased from P6 to P18 (Fig. 5 C). The area of nerve terminal was 67.16 ± 4.52 μm2 (n = 41, N =5) at P6, 84.35 ± 3.35 μm2 (n = 75, N 5) at P10 and 174.93 ± 5.81 μm2 (n = 119, N = 5) at Р18 animals (Fig. 5 C). These data are consistent with results obtained earlier where specific antibodies to the presynaptic protein synaptophysin were used as marker of the presynaptic area (Khuzakhmetova et al., 2014a). In pups with hHCY the average area of presynaptic nerve terminals at P6 and P10 animals was larger compared to controls (86.05 ± 5.62 μm2, n = 45, N = 5, p < 0.05 and 111.13 ± 3.05 μm2, n =118, N =5 respectively; p < 0.05) (Fig. 5 C). However, at P18 the area of nerve terminals was less than in control rats (120.81 ± 3.56 μm2, n = 120, N = 5, p < 0.05, Fig. 5 C). The analysis of terminal staining revealed an increase of fluorescence intensity by the third week of development from 99.7 ± 4.5 a.u. (n = 35, N = 5, Table S2) at P6 to 116.2 ± 3.3 a.u. at P18 (n =89, N=5). The intensity of nerve terminals staining from hHCY rats of all aged groups was not different from control values (Table S2). 11.

(14) IP T SC R U N A M ED PT. A. CC E. Fig. 5. FM1-43 staining of motor terminals in rat neuromuscular synapses. (A) Typical images of presynaptic nerve terminals of P6, P10 and P18 rats loaded with FM1-43. (B) The percentage of different type of synapse in control and hHCY animals. White color – type 1 synapses, grey - type 2 and black – type 3. (C) Average area of nerve terminals at P6, P10 and P18 rats of control and hHCY groups. Boxes indicate 25–75 percentiles, black line—median, whiskers—minimal and maximal values. In control: P6 – n = 41, N = 5; P10 – n = 75, N = 5; P18 – n = 119, N = 5. In hHCY: P6 – n = 45, N = 5; P10 – n = 118, N = 5; P18 – n = 120, N = 5. *p < 0.05 - compared to control group.. 3.7 Muscle strength Muscle strength was assessed using the PaGE test, where the time to fall from the grid was measured in neonatal and adult rats. A significant reduction of time spent on the grid was 12.

(15) observed in the hHCY P10 and adult animals compared to control. At P10 in the control group the time rats were able to stay on the grid was 6.06±0.95 s (N = 25) and in hHCY group - 4.14 ± 0.35 s (N = 25, p < 0.05). In adult rats we also observed a deficit in the PaGE task (75.80 ± 12.36 s, N = 25 in control vs. 43.64 ± 8.59 s, N = 24, p < 0.05 in hHCY). 3.8 ROS level and antioxidant enzyme activity in diaphragm muscles of rats from control and hHCY groups. A. CC E. PT. ED. M. A. N. U. SC R. IP T. In order to assess the level of oxidative stress induced by hHCY we measured ROS production and the activity of antioxidant enzymes in diaphragm muscles of rats from hHCY and control groups. We found that the level of endogenous peroxides was higher in diaphragm muscles of hHCY rats of all ages compared to controls (Fig. 6 A). The data reflect the ability of HCY to stimulate intracellular superoxide radical production (Veeranki and Tyagi, 2013). It is known that high levels of ROS can induce lipid peroxidation damaging cell membrane integrity (Poon et al., 2004). Indeed, in muscles of P6, P10 and adults hHCY rats we observed an increased formation of MDA, an end product of lipid peroxidation (Fig. 6 B). Oxidative stress induced by HCY may be aggravated by the impairment of antioxidant enzyme activity. To address this issue, we examined enzymatic activities of SOD, catalase and GPx in muscles from control and hHCY groups. We found that SOD activity, that converts superoxide anions into H2O2, was significantly decreased in neonatal and adult hHCY animals (Fig. 6 C). In contrary, activity of catalase, which converts H2O2 to water and oxygen, was increased in neonatal and adult hHCY groups (Fig. 6 D). A decreased activity of GPx, which reduces peroxides, was observed in all groups of hHCY animals (Fig. 6 E). These alterations in the redox balance of key antioxidant enzymes and increased ROS level in the skeletal muscles of hHCY rats may contribute to the changes of synaptic transmission in the peripheral synapses and the vulnerability of nerve terminals to exogenous ROS which we observed in our functional tests.. 13.

(16) IP T SC R U N A M ED PT CC E A. Fig. 6. Effects of prenatal hHCY on ROS level and antioxidant enzyme activities measured in neonatal and adult rat diaphragm muscles. The level of peroxides (A) and MDA - an end product of lipoperoxidation (B) in diaphragm muscles of P6, P10 and adult rats from control and hHCY groups. Activities of the enzymes, superoxide dismutase 1 (C), catalase (D) and glutathione peroxidase 1 (E) measured in cytosolic fractions of diaphragm muscles derived from P6, P10 and adult rats from control and hHCY groups. In control: P6 –N = 7; P10 – N = 7; adult – N = 6. In hHCY: P6 – N = 6, P10 – N = 7, adult – N = 10. *p<0.05 – compared to control group.. 4. DISCUSSION 14.

(17) IP T. In our study we present for the first time changes of the synaptic transmission during postnatal development in rat neuromuscular junction caused by elevated levels of the endogenous amino acid HCY during the prenatal period. Several previous studies reported that an increased level of HCY leads to functional impairments and weakness of skeletal muscle (Veeranki and Tyagi, 2013; Ng et al., 2012; Swart et al., 2013; Majumder et al., 2017, 2018a,b). Children born with severe homocystinuria due to CBS deficiency exhibit low body weight and skeletal muscle myopathy (Picker and Levy, 2010). However, the effects of chronic maternal hHCY on peripheral synaptic transmission and redox status in skeletal muscles were not investigated. In the present study we show how the transmitter release from the motor nerve endings, synapse morphology, muscle strength and redox status are modified in animals with prenatal hHCY. In our model of prenatal methionine-based dietary hHCY the level of HCY is elevated not only in dams but also in their offspring suggesting an attractive approach to test the role of HCY in early development (Gerasimova et al., 2017; Yakovleva et al., 2018).. SC R. 4.1 Faster maturation of presynaptic area in synapses of neonatal rats with maternal hHCY. CC E. PT. ED. M. A. N. U. The mammalian neuromuscular junction undergoes dramatic changes in structure and functions during the first few postnatal weeks. We assessed the shape and area of presynaptic nerve endings at P6, P10 and adult rats using the fluorescence dye FM 1-43 which was loaded into the terminals during high frequency stimulation (Petrov et al., 2010; Yakovleva et al., 2017). We observed three types of synaptic contacts which developed dependent on the animal age. In control at P6 plaque-like nerve terminals with uniform fluorescence were observed reflecting the absence of discrete active zones as was shown earlier (Linden et al., 1988). Later they were replaced by branched terminals at P10 and finally by multi-perforated adult-like synapses (a pretzel-like shape) at P18, where discrete fluorescence spots indicated the clusters of synaptic vesicles in active zones (Dennis et al., 1981; Sanes and Lichtman, 1999). The average area of presynaptic nerve terminals increased with age, respectively. However, in animals with prenatal hHCY mature forms of presynaptic terminals were found already during the first week of development and also the average area of synaptic contacts was larger at P6 and P10 animals compared to controls. We suggested that faster maturation of presynaptic terminals in hHCY rats may be induced by activation of protein kinase C (PKC) (Loureiro et al., 2008; Signorello et al., 2009) which is important for maturation and maintenance of neuromuscular junctions (Lanuza et al., 2002). At the same time at P18 the number of adult-like synapses and the area of presynaptic terminals were smaller compared to control, which correspond to synapses of aged animals (Gonzalez-Freire et al., 2014; Jang and Van Remmen, 2011). The fluorescence analysis of nerve terminals did not reveal differences between the two groups of animals indicating that the intensity of endocytosis of synaptic vesicles was not changed at least with the protocol of FM 143 loading used in this study.. A. 4.2 The intensity of transmitter release in synapses of neonatal and adult rats with prenatal hHCY In our experiments, we found an increased amplitude and faster time course of spontaneous and evoked uni-quantal EPCs in neonates with hHCY. These data may indicate the early replacement of the fetal ε-subunits of acetylcholine receptors by γ-subunits characterized by a higher conductance and shorter open time (Missias et al., 1996; Witzemann, 2006). Along with postsynaptic changes, concomitant changes were observed in the probability and timing of quantal release in hHCY rats. In control MEPCs frequency progressively grows with animal maturation (Diamond and Miledi, 1962; Shakirzyanova et al., 2016). However, in hHCY, a higher intensity of spontaneous release was already observed at P10 animals. Likewise, in the 15.

(18) synapses of newborns with hHCY evoked acetylcholine release was significantly higher compared to controls and did not change with aging and therefore was lower in adults compared to the control group probably due to associated oxidative stress (Gonzalez-Freire et al., 2014; Jang and Van Remmen, 2011). 4.3 The timing of transmitter release in synapses of neonatal and adult rats with prenatal hHCY. CC E. PT. ED. M. A. N. U. SC R. IP T. The minimal synaptic delays assessed from uni-quantal EPCs characterize the most ready to release quanta (Barrett and Stevens, 1972; Bukcharaeva et al., 1999; Katz and Miledi, 1965). During maturation a progressive decrease of minimal synaptic delays along with synchronization of quantal release are observed (Khuzakhmetova et al., 2014a). Indeed, in our experiments at the synapses of P6 and P10 control animals a low probability of release was accompanied by longer minimal synaptic delays and substantial asynchrony of release when most quanta were released during late synchronous and delayed asynchronous periods. In neonates from the hHCY group the minimal synaptic delays were shorter compared to control and achieved the values of adult animals. In addition, the quantal release in synapses of hHCY newborns was highly synchronized. These data are consistent with a higher release probability in hHCY neonates. High level of asynchrony in newborns observed in central and peripheral synapses are mainly explained by the poor maturity of the mechanisms controlling the intracellular Ca2+ concentration (Chang and Mennerick, 2010). In fact, developmental upregulation of endogenous Ca2+ buffering proteins, such as calretinin and parvalbumin, low Ca2+ affinity of synaptotagmins and reduced efficiency of Ca2+ buffers were demonstrated in immature synapses (Iwasaki et al., 2000; Chuhma et al., 2001; Gilmanov et al., 2008). In addition, during early stages of ontogenesis along with P/Q type Ca2+ channels, L-type Ca channels with slower kinetics and a more distant location to active zones are involved in transmitter release (Nudler et al., 2003; Khuzakhmetova, 2016). The faster kinetics and higher synchronization of quantal release in hHCY could be explained by faster maturation of the above mentioned mechanisms and higher probability of release due to a more effective increase of intracellular Ca2+ which involves activation of voltage-dependent Ca2+ channels, as well as Ca2+ release from intracellular stores (Loureiro et al., 2008). High intracellular Ca2+ not only increases the probability of release, but also contributes to its synchronization (Dodge and Rahamimoff, 1967; Bukharaeva et al., 2007). In contrast, at the synapses of adults from the hHCY group the number of quanta released during phasic period was lower along with less probability of release compared to control which may indicate aging processes. Acute application of exogenous HCY at the neuromuscular junction did not reveal significant changes in the amplitude and temporal parameters of MEPCs and EPCs in all groups of animals. These results are in agreement with our previous observation where short-term application of HCY did not affect MEPP frequency and an altered activity of ion channels by itself (Bukharaeva et al., 2015; Gaifullina et al., 2016).. A. 4.4 Susceptibility of synapses of neonatal rats with prenatal hHCY to H2O2 In a previous study we have shown that HCY amplified the inhibitory effects of H2O2 on spontaneous acetylcholine release in mouse neuromuscular junction through activation of NMDA receptors (Bukharaeva et al., 2015). Recently, these findings were supported by Wang et al. (2018), where the involvement of nitric oxide in the sensitizing effect of HCY to ROS was revealed. Therefore, we compared the action of H2O2 at the synapses of control and hHCY rats. Consistent with our previous data (Shakirzyanova et al., 2016), in control, H2O2 caused a significant decrease in the frequency of MEPCs in adult animals without any effects in newborn rats. Interestingly, in animals with prenatal hHCY the inhibitory effect of H2O2 was already observed in neonates. However, we did not see the effects of H2O2 on evoked transmitter release 16.

(19) 4.5 hHCY induced the muscle weakness in neonatal and adult rats. IP T. as in our previous study (Shakirzyanova et al., 2016), which may be explained by the low external Ca2+ concentration used in the present experimental approach. Nevertheless, we found that H2O2 synchronized the quantal release in neonates from the hHCY group which may result from the prolonged Ca2+ influx engaging distant synaptic vesicles (Giniatullin and Giniatullin, 2003). The neonatal resistance of spontaneous transmitter release to ROS appeared to result from immature mechanisms of intracellular Ca2+ control (Shakirzyanova et al., 2016). The ability of HCY to increase intraterminal Ca2+ (Loureiro et al., 2008; Robert et al., 2005) may underlie the higher sensitivity of spontaneous release to H2O2 in animals with hHCY. Moreover, HCY induced activation of protein kinase C (PKC) (Loureiro et al., 2008; Signorello et al., 2009) which restores the redox sensitivity of MEPPs in newborns (Shakirzyanova et al., 2016).. U. SC R. The Paw Grip Endurance (PaGE) test demonstrated that in both groups (P10 and adults) the time spent on the grid was lower in hHCY rats indicating diminished muscle strength. These data are consistent with previously obtained data, where hHCY exerted detrimental effects on muscle force generation and fatigability (Veeranki et al., 2015; Majumder et al., 2018b; Yakovleva et al., 2018). The molecular mechanisms of detrimental effects of hHCY include mitochondrial dysfunctions, oxidative/endoplasmic reticulum (ER)-stress and secretion of pro-inflammatory cytokines (Veeranki et al., 2015; Majumder et al., 2018a).. N. 4.6 The level of oxidative stress in diaphragm muscles of rats with maternal hHCY. A. CC E. PT. ED. M. A. Oxidative stress plays an important role in the effects of hHCY, promoting apoptosis, inflammation, insulin resistance, skeletal muscle damage and dysregulation of lipid metabolism (DiBello et al., 2010; Veeranki and Tyagi, 2013; Majumder et al., 2018a). Indeed, using a spectrophotometric assay we found an increase of H2O2 level and lipid peroxidation in muscles of neonatal and adult rats in the hHCY group. Apparently, this increased level of ROS can cause oxidation of various functionally important proteins, including the presynaptic transmitter releasing machinery which is the most redox-sensitive part of the motor synapse (Giniatullin et al., 2006; Majumder et al., 2017; Veeranki and Tyagi, 2013) and as a result causes the functional changes of neuromuscular synapses in hHCY. The level of oxidative stress in hHCY animals was aggravated by the decreased activity of two endogenous antioxidants - SOD and GPx. At the same time catalase activity was increased in P6 and adult animals. Previous data indicate that the changes of antioxidant enzyme activity depend on the tissue type and/or acute or chronic treatment with HCY. Thus, in the brain of animals with prenatal hHCY SOD activity was decreased (Makhro et al., 2008). In soleus skeletal muscle of rats SOD and CAT activity were increased, but GPx activity was not altered (Kolling et al., 2014). The decreased expression and activity of GPx-1 induced by HCY was shown in vitro and in vivo studies (Lubos et al., 2007; Outinen et al., 1999). The enhanced catalase activity may be a consequence of tissue adaptation to the sustained production of ROS (Kolling et al., 2014), but cannot overcome the increasing oxidative stress. This imbalance in the activity of antioxidant enzymes caused by HCY may endow the alteration of peripheral synaptic transmission in animals with prenatal hHCY. Redox signaling is important in developmental processes controlling proliferation, apoptosis, neuronal differentiation and axon formation. The redox status is tightly regulated during development (Marseglia et al., 2014; Timme-Laragy et al., 2018). Oxidative stress causes synapse loss as a result of impaired mitochondrial function (Mast et al., 2008). On the other hand oxidative stress may result in synapse overgrowth and faster maturation as a response to activation of Jun-N-terminal kinase (JNK) signaling which regulates synapse size and strength through phosphorylation of the activator protein 1 (AP-1) (Sanyal et al., 2002; Kim et al., 2009). 17.

(20) Our study has few limitations. One limitation in that electrophysiological experiments were performed in low extracellular Ca2+, which nevertheless allowed us to assess more precisely the quantum content of EPCs and the level of synchronicity of release which was shown to be changed considerably from neonatal to adult age (Khuzachmetova et al., 2014a). Second limitation that we did not used old rats with hHCY in our experiments, which may reveal more clearly the disturbances of peripheral synaptic transmission in aged animals. Finally, we did not study in detail the processes of synaptic vesicles recycling in motor nerve endings, which may disclose other age-dependent differences in control and hHCY animals. Future studies using antioxidants during pregnancy and neonatal period would be helpful in developing strategies to prevent the disturbances of synaptic transmission during hHCY conditions.. N. U. SC R. IP T. 5. Conclusion Prenatal hHCY induces oxidative stress which is as a fundamental component of aging, a process that begins before birth (Marseglia et al., 2014; Buonocore et al., 2011; Gonzalez-Freire et al., 2014). This results in faster maturation of synapses in newborn animals with hHCY - but also to accelerated aging in adults. Indeed, several human studies have shown that hHCY aggravates the age-associated decline of motor functions, causing skeletal muscle injury and elderly frailty, associated with reduced physical performance and muscle strength in elderly persons (Ng et al., 2012; Swart et al., 2013). Elevated levels of HCY are associated with ALS (Valentino et al., 2010; Zoccolella et al., 2012). Apparently, the impairments of synaptic transmission in peripheral synapses could be one of the mechanisms underlying the compromised muscle function observed in hHCY patients during aging and in motor neuronal disease like ALS and multiple sclerosis (Naumenko et al., 2011; Pollari et al., 2011).. M. A. Data Availability The data used to support the findings of this study are available from the corresponding author upon request.. ED. Conflict of interest Acknowledgments The authors declare no conflict of interests regarding this publication.. A. CC E. PT. Acknowledgments. The authors are grateful to Anton Hermann, University of Salzburg for useful suggestions. This project was supported by the Russian Science Foundation (Grant No. 14-15-00618) and was performed in accordance with the Program of Competitive Growth of Kazan Federal University. VK and EB were supported by budget financing from RAS.. 18.

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