Myostatin Inhibition-Induced Increase in Muscle Mass and Strength Was Amplified by Resistance Exercise Training, and Dietary Essential Amino Acids Improved Muscle Quality in Mice.

It has been frequently reported that myostatin inhibition increases muscle mass, but decreases muscle quality (i.e., strength/muscle mass). Resistance exercise training (RT) and essential amino acids (EAAs) are potent anabolic stimuli that synergistically increase muscle mass through changes in muscle protein turnover. In addition, EAAs are known to stimulate mitochondrial biogenesis. We have investigated if RT amplifies the anabolic potential of myostatin inhibition while EAAs enhance muscle quality through stimulations of mitochondrial biogenesis and/or muscle protein turnover. Mice were assigned into ACV (myostatin inhibitor), ACV+EAA, ACV+RT, ACV+EAA +RT, or control (CON) over 4 weeks. RT, but not EAA, increased muscle mass above ACV. Despite differences in muscle mass gain, myofibrillar protein synthesis was stimulated similarly in all vs. CON, suggesting a role for changes in protein breakdown in muscle mass gains. There were increases in MyoD expression but decreases in Atrogin-1/MAFbx expression in ACV+EAA, ACV+RT, and ACV+EAA+RT vs. CON. EAA increased muscle quality (e.g., grip strength and maximal carrying load) without corresponding changes in markers of mitochondrial biogenesis and neuromuscular junction stability. In conclusion, RT amplifies muscle mass and strength through changes in muscle protein turnover in conjunction with changes in implicated signaling, while EAAs enhance muscle quality through unknown mechanisms.

Structural and biochemical analyses reveal ubiquitin C-terminal hydrolase-L1 as a specific client of the peroxiredoxin II chaperone.

Peroxiredoxins (Prxs) play dual roles as both thiol-peroxidases and molecular chaperones. Peroxidase activity enables various intracellular functions, however, the physiological roles of Prxs as chaperones are not well established. To study the chaperoning function of Prx, we previously sought to identify heat-induced Prx-binding proteins as the clients of a Prx chaperone. By using His-tagged Prx I as a bait, we separated ubiquitin C-terminal hydrolase-L1 (UCH-L1) as a heat-induced Prx I binding protein from rat brain crude extracts. Protein complex immunoprecipitation with HeLa cell lysates revealed that both Prx I and Prx II interact with UCH-L1. However, Prx II interacted considerably more favorably with UCH-L1 than Prx I. Prx II exhibited more effective molecular chaperone activity than Prx I when UCH-L1 was the client. Prx II interacted with UCH-L1 through its C-terminal region to protect UCH-L1 from thermal or oxidative inactivation. We found that chaperoning via interaction through C-terminal region (specific-client chaperoning) is more efficient than that involving oligomeric structural change (general-client chaperoning). Prx II binds either thermally or oxidatively unfolding early intermediates of specific clients and thereby shifted the equilibrium towards their native state. We conclude that this chaperoning mechanism provides a very effective and selective chaperoning activity.

Methods for chronic recording of EMG activity from large numbers of hindlimb muscles in awake rhesus macaques.

Studies of the neural control of movement often rely on the ability to record EMG activity during natural behavioral tasks over long periods of time. Increasing the number of recorded muscles and the time over which recordings are made allows more rigorous answers to many questions related to the descending control of motor output. Chronic recording of EMG activity from multiple hindlimb muscles has been reported in the cat but few studies have been done in non-human primates. This paper describes two chronic EMG implant methods that are minimally invasive, relatively non-traumatic and capable of recording from large numbers of hindlimb muscles simultaneously for periods of many months to years.

Forelimb muscle representations and output properties of motor areas in the mesial wall of rhesus macaques.

In this study, forelimb organizations and output properties of the supplementary motor area (SMA) and the dorsal cingulate motor area (CMAd) were assessed and compared with primary motor cortex (M1). Stimulus-triggered averages of electromyographic activity from 24 muscles of the forelimb were computed from layer V sites of 2 rhesus monkeys performing a reach-to-grasp task. No clear segregation of the forelimb representation of proximal and distal muscles was found in SMA. In CMAd, sites producing poststimulus effects in proximal muscles tended to be located caudal to distal muscle sites, although the number of effects was limited. For both SMA and CMAd, facilitation effects were more prevalent in distal than in proximal muscles. At an intensity of 60 microA, the mean latencies of M1 facilitation effects were 8 and 12.1 ms shorter and the magnitudes approximately 10 times greater than those from SMA and CMAd. Our results show that corticospinal neurons in SMA and CMAd provide relatively weak input to spinal motoneurons compared with the robust effects from M1. However, a small number of facilitation effects from SMA and CMAd had latencies as short as the shortest ones from M1 suggesting a minimum linkage to motoneurons as direct as that from M1.

In vivo detection of gray and white matter differences in GABA concentration in the human brain.

A novel selective multiple quantum filtering-based chemical shift imaging method was developed for acquiring GABA images in the human brain at 3 T. This method allows a concomitant acquisition of an interleaved total creatine image with the same spatial resolution. Using T(1)-based image segmentation and a nonlinear least square regression analysis of GABA-to-total creatine concentration ratios in frontal and parietal lobes of healthy adult volunteers as a function of the tissue gray matter fraction, the mean GABA concentration in gray and white matter was determined to be 1.30+/-0.36 micromol/g and 0.16+/-0.16 micromol/g (mean+/-SD, n=13), respectively. It is expected that this method will become a useful tool for studying GABAergic function in the human brain in vivo.

Magnetic field correlation imaging.

A magnetic resonance imaging (MRI) method is presented for estimating the magnetic field correlation (MFC) associated with magnetic field inhomogeneities (MFIs) within biological tissues. The method utilizes asymmetric spin echoes and is based on a detailed theory for the effect of MFIs on nuclear magnetic resonance (NMR) signal decay. The validity of the method is supported with results from phantom experiments at 1.5 and 3 T, and human brain images obtained at 3 T are shown to demonstrate the method's feasibility. The preliminary results suggest that MFC imaging may be useful for the quantitative assessment of iron within the brain.

Redox-regulated cochaperone activity of the human DnaJ homolog Hdj2.

The human DnaJ homolog Hdj2 is a cochaperone containing a cysteine-rich zinc finger domain. We identified a specific interaction of Hdj2 with the cellular redox enzyme thioredoxin using a yeast two-hybrid assay and a coimmunoprecipitation assay, thereby investigating how the redox environment of the cell regulates Hdj2 function. In reconstitution experiments with Hsc70, we found that treatment with H2O2 caused the oxidative inactivation of Hdj2 cochaperone activity. Hdj2 inactivation paralleled the oxidation of cysteine thiols and concomitant release of coordinated zinc, suggesting a role of cysteine residues in the zinc finger domain of Hdj2 as a redox sensor of chaperone-mediated protein-folding machinery. H2O2-induced negative regulation of Hdj2 cochaperone activity was also confirmed in mammalian cells using luciferase as a foreign reporter cotransfected with Hsc70 and Hdj2. The in vivo oxidation of cysteine residues in Hdj2 was detected only in thioredoxin-knockdown cells, implying that thioredoxin is involved in the in vivo reduction. The oxidative inactivation of Hdj2 was reversible. Wild-type thioredoxin notably recovered the oxidatively inactivated Hdj2 activity accompanied by the reincorporation of zinc, whereas the catalytically inactive mutant thioredoxin (Cys32Ser/Cys35Ser) did not. Taken together, we propose that oxidation and reduction reversibly regulate Hdj2 function in response to the redox states of the cell.

Histological co-localization of iron in Abeta plaques of PS/APP transgenic mice.

This study confirms the presence of iron, co-localized with Abeta plaques, in PS/APP mouse brain, using Perls' stain for Fe3+ supplemented by 3,3'-diaminobenzidine (DAB) and Abeta immunohistochemistry in histological brains sections fixed with formalin or methacarn. In this study, the fixation process and the slice thickness did not interfere with the Perls' technique. The presence of iron in beta-amyloid plaques in PS/APP transgenic mice, a model of Alzheimer's disease (AD) pathology, may explain previous reports of reductions of transverse relaxation time (T2) in MRI studies and represent the source of the intrinsic Abeta plaque MR contrast in this model.

Selective homonuclear Hartmann-Hahn transfer method for in vivo spectral editing in the human brain.

A novel selective homonuclear Hartmann-Hahn transfer method for in vivo spectral editing is proposed and applied to measurements of gamma-aminobutyric acid (GABA) in the human brain at 3 T. The proposed method utilizes a new concept for in vivo spectral editing, the spectral selectivity of which is not based on a conventional editing pulse but based on the stringent requirement of the doubly selective Hartmann-Hahn match. The sensitivity and spectral selectivity of GABA detection achieved by this doubly selective Hartmann-Hahn match scheme was superior to that achievable by conventional in vivo spectral editing techniques providing both sensitivity enhancement and excellent suppression of overlapping resonances in a single shot. Since double-quantum filtering gradients were not employed, singlets such as the NAA methyl group at 2.02 ppm and the creatine methylene group at 3.92 ppm were detected simultaneously. These singlets may serve as navigators for the spectral phase of GABA and for frequency shifts during measurements. The estimated concentration of GABA in the frontoparietal region of the human brain in vivo was 0.7 +/- 0.2 mumol/g (mean +/- SD, n = 12).

In vivo single-shot three-dimensionally localized multiple quantum spectroscopy of GABA in the human brain with improved spectral selectivity.

A single-shot multiple quantum filtering method is developed that uses two double-band frequency selective pulses for enhanced spectral selectivity in combination with a slice-selective 90 degrees, a slice-selective universal rotator 90 degrees, and a spectral-spatial pulse composed of two slice-selective universal rotator 45 degrees pulses for single-shot three-dimensional localization. The use of this selective multiple quantum filtering method for C(3) and C(4) methylene protons of GABA resulted in improved spectral selectivity for GABA and effective suppression of overlapping signals such as creatine and glutathione in each single scan, providing reliable measurements of the GABA doublet in all subjects. The concentration of GABA was measured to be 0.7 +/- 0.2 micromol/g (means +/- SD, n = 15) in the fronto-parietal region of the human brain in vivo.

Visualization of beta-amyloid plaques in a transgenic mouse model of Alzheimer's disease using MR microscopy without contrast reagents.

The visualization of beta-amyloid plaque deposition in brain, a key feature of Alzheimer's disease (AD), is important for the evaluation of disease progression and the efficacy of therapeutic interventions. In this study, beta-amyloid plaques in the PS/APP transgenic mouse brain, a model of human AD pathology, were detected using MR microscopy without contrast reagents. beta-Amyloid plaques were clearly visible in the cortex, thalamus, and hippocampus of fixed brains of PS/APP mice. The distribution of plaques identified by MRI was in excellent agreement with those found in the immunohistological analysis of the same brain sections. It was also demonstrated that image contrast for beta-amyloid plaques was present in freshly excised nonfixed brains. Furthermore, the detection of beta-amyloid plaques was achieved with a scan time as short as 2 hr, approaching the scan time considered reasonable for in vivo imaging.

Quantitative MRI assessment of Alzheimer's disease.

The development of a noninvasive method to detect early, subtle changes in the brains of patients with Alzheimer's disease (AD) would have considerable clinical value as therapy. This therapy is most likely to be successful if intervention could occur before neurons were irreversibly damaged or lost. An ideal biological neuroimaging marker would be an early, sensitive, and valid indicator of brain changes, capable of discriminating the effects of normal aging. The introduction of high field-strength clinical magnetic resonance imaging (MRI) systems now offer a powerful new noninvasive tool that may be capable of detecting brain pathology resulting from AD. Here we present results from high field-strength MRI in transgenic mice along with a new MRI technique for imaging brain iron. The successful translation of this research to the clinic could prove important to both the early diagnosis and monitoring of the efficacy of potential therapies in humans.

Single-shot two-echo technique for simultaneous measurement of GABA and creatine in the human brain in vivo.

A single-shot, two-echo method for the simultaneous detection of multiple-quantum (MQ)-filtered gamma-aminobutyric acid (GABA) and creatine (Cr) was developed and demonstrated in the human brain in vivo at 3 Tesla. The simultaneously measured Cr singlet served as a navigator for the spectral phase of GABA and any frequency shift during measurements due to drift in the static magnetic field (B(0)) or subject movement, as well as an internal concentration reference. In addition, the use of a double-band frequency-selective MQ filter for C(3) and C(4) methylene protons of GABA provided a very robust measurement of GABA, with excellent suppression of overlapping metabolites such as Cr and glutathione (GSH) in each single scan. Contamination from overlapping macromolecules was also demonstrated to be negligible with this method. The GABA-to-Cr ratio was 0.09 +/- 0.03 (mean +/- SD, N = 17) and the estimated concentration of GABA in the frontoparietal region of the human brain in vivo was 0.66 +/- 0.19 micromol/g (mean +/- SD, N = 17) with the internal reference method, and 0.69 +/- 0.18 micromol/g (mean +/- SD, N = 17) with the external reference method. The observed pattern of GABA doublet was consistent among all subjects, with a frequency separation of approximately 13 Hz.

MRI assessment of neuropathology in a transgenic mouse model of Alzheimer's disease.

The cerebral deposition of amyloid beta-peptide, a central event in Alzheimer's disease (AD) pathogenesis, begins several years before the onset of clinical symptoms. Noninvasive detection of AD pathology at this initial stage would facilitate intervention and enhance treatment success. In this study, high-field MRI was used to detect changes in regional brain MR relaxation times in three types of mice: 1). transgenic mice (PS/APP) carrying both mutant genes for amyloid precursor protein (APP) and presenilin (PS), which have high levels and clear accumulation of beta-amyloid in several brain regions, starting from 10 weeks of age; 2). transgenic mice (PS) carrying only a mutant gene for presenilin (PS), which show subtly elevated levels of Abeta-peptide without beta-amyloid deposition; and 3). nontransgenic (NTg) littermates as controls. The transverse relaxation time T(2), an intrinsic MR parameter thought to reflect impaired cell physiology, was significantly reduced in the hippocampus, cingulate, and retrosplenial cortex, but not the corpus callosum, of PS-APP mice compared to NTg. No differences in T(1) values or proton density were detected between any groups of mice. These results indicate that T(2) may be a sensitive marker of abnormalities in this transgenic mouse model of AD.

In vivo NMR studies of neurodegenerative diseases in transgenic and rodent models.

In vivo magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) provide unique quality to attain neurochemical, physiological, anatomical, and functional information non-invasively. These techniques have been increasingly applied to biomedical research and clinical usage in diagnosis and prognosis of diseases. The ability of MRS to detect early yet subtle changes of neurochemicals in vivo permits the use of this technology for the study of cerebral metabolism in physiological and pathological conditions. Recent advances in MR technology have further extended its use to assess the etiology and progression of neurodegeneration. This review focuses on the current technical advances and the applications of MRS and MRI in the study of neurodegenerative disease animal models including amyotrophic lateral sclerosis, Alzheimer's, Huntington's, and Parkinson's diseases. Enhanced MR measurable neurochemical parameters in vivo are described in regard to their importance in neurodegenerative disorders and their investigation into the metabolic alterations accompanying the pathogenesis of neurodegeneration.

Origin of negative blood oxygenation level-dependent fMRI signals.

Functional magnetic resonance imaging (fMRI) techniques are based on the assumption that changes in spike activity are accompanied by modulation in the blood oxygenation level-dependent (BOLD) signal. In addition to conventional increases in BOLD signals, sustained negative BOLD signal changes are occasionally observed and are thought to reflect a decrease in neural activity. In this study, the source of the negative BOLD signal was investigated using T2*-weighted BOLD and cerebral blood volume (CBV) techniques in isoflurane-anesthetized cats. A positive BOLD signal change was observed in the primary visual cortex (area 18) during visual stimulation, while a prolonged negative BOLD change was detected in the adjacent suprasylvian gyrus containing higher-order visual areas. However, in both regions neurons are known to increase spike activity during visual stimulation. The positive and negative BOLD amplitudes obtained at six spatial-frequency stimuli were highly correlated, and negative BOLD percent changes were approximately one third of the positive changes. Area 18 with positive BOLD signals experienced an increase in CBV, while regions exhibiting the prolonged negative BOLD signal underwent a decrease in CBV. The CBV changes in area 18 were faster than the BOLD signals from the same corresponding region and the CBV changes in the suprasylvian gyrus. The results support the notion that reallocation of cortical blood resources could overcome a local demand for increased cerebral blood flow induced by increased neural activity. The findings of this study imply that caution should be taken when interpreting the negative BOLD signals as a decrease in neuronal activity.

Comparison of diffusion-weighted high-resolution CBF and spin-echo BOLD fMRI at 9.4 T.

The quantification of blood oxygenation-level dependent (BOLD) functional MRI (fMRI) signals is closely related to cerebral blood flow (CBF) change; therefore, understanding the exact relationship between BOLD and CBF changes on a pixel-by-pixel basis is fundamental. In this study, quantitative CBF changes induced by neural activity were used to quantify BOLD signal changes during somatosensory stimulation in alpha-chloralose-anesthetized rats. To examine the influence of fast-moving vascular spins in quantifying CBF, bipolar gradients were employed. Our data show no significant difference in relative CBF changes obtained with and without bipolar gradients. To compare BOLD and CBF signal changes induced by neural stimulation, a spin-echo (SE) sequence with long SE time of 40 ms at 9.4 T was used in conjunction with an arterial spin labeling technique. SE BOLD changes were quantitatively correlated to CBF changes on a pixel-by-pixel and animal-by-animal basis. Thus, SE BOLD-based fMRI at high magnetic fields allows a quantitative comparison of functional brain activities across brain regions and subjects.