α-Ketoglutarate supplementation and NAD+ modulation enhance metabolic rewiring and radiosensitization in SLC25A1 inhibited cancer cells.

Metabolic rewiring is the result of the increasing demands and proliferation of cancer cells, leading to changes in the biological activities and responses to treatment of cancer cells. The mitochondrial citrate transport protein SLC25A1 is involved in metabolic reprogramming offering a strategy to induce metabolic bottlenecks relevant to radiosensitization through the accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) upon SLC25A1 inhibition (SLC25A1i). Previous studies have revealed the comparative effects of SLC25A1i or cell-permeable D-2HG (octyl-D-2HG) treatments on DNA damage induction and repair, as well as on energy metabolism and cellular function, which are crucial for the long-term survival of irradiated cells. Here, α-ketoglutarate (αKG), the precursor of D-2HG, potentiated the effects observed upon SLC25A1i on DNA damage repair, cell function and long-term survival in vitro and in vivo, rendering NCI-H460 cancer cells more vulnerable to ionizing radiation. However, αKG treatment alone had little effect on these phenotypes. In addition, supplementation with nicotinamide (NAM), a precursor of NAD (including NAD+ and NADH), counteracted the effects of SLC25A1i or the combination of SLC25A1i with αKG, highlighting a potential importance of the NAD+/NADH balance on cellular activities relevant to the survival of irradiated cancer cells upon SLC25A1i. Furthermore, inhibition of histone lysine demethylases (KDMs), as a major factor affected upon SLC25A1i, by JIB04 treatment alone or in combination with αKG supplementation phenocopied the broad effects on mitochondrial and cellular function induced by SLC25A1i. Taken together, αKG supplementation potentiated the effects on cellular processes observed upon SLC25A1i and increased the cellular demand for NAD to rebalance the cellular state and ensure survival after irradiation. Future studies will elucidate the underlying metabolic reprogramming induced by SLC25A1i and provide novel therapeutic strategies for cancer treatment.

Targeting AKT-Dependent Regulation of Antioxidant Defense Sensitizes AKT-E17K Expressing Cancer Cells to Ionizing Radiation.

Aberrant activation of the phosphatidyl-inositol-3-kinase/protein kinase B (AKT) pathway has clinical relevance to radiation resistance, but the underlying mechanisms are incompletely understood. Protection against reactive oxygen species (ROS) plays an emerging role in the regulation of cell survival upon irradiation. AKT-dependent signaling participates in the regulation of cellular antioxidant defense. Here, we were interested to explore a yet unknown role of aberrant activation of AKT in regulating antioxidant defense in response to IR and associated radiation resistance. We combined genetic and pharmacologic approaches to study how aberrant activation of AKT impacts cell metabolism, antioxidant defense, and radiosensitivity. Therefore, we used TRAMPC1 (TrC1) prostate cancer cells overexpressing the clinically relevant AKT-variant AKT-E17K with increased AKT activity or wildtype AKT (AKT-WT) and analyzed the consequences of direct AKT inhibition (MK2206) and inhibition of AKT-dependent metabolic enzymes on the levels of cellular ROS, antioxidant capacity, metabolic state, short-term and long-term survival without and with irradiation. TrC1 cells expressing the clinically relevant AKT1-E17K variant were characterized by improved antioxidant defense compared to TrC1 AKT-WT cells and this was associated with increased radiation resistance. The underlying mechanisms involved AKT-dependent direct and indirect regulation of cellular levels of reduced glutathione (GSH). Pharmacologic inhibition of specific AKT-dependent metabolic enzymes supporting defense against oxidative stress, e.g., inhibition of glutathione synthase and glutathione reductase, improved eradication of clonogenic tumor cells, particularly of TrC1 cells overexpressing AKT-E17K. We conclude that improved capacity of TrC1 AKT-E17K cells to balance antioxidant defense with provision of energy and other metabolites upon irradiation compared to TrC1 AKT-WT cells contributes to their increased radiation resistance. Our findings on the importance of glutathione de novo synthesis and glutathione regeneration for radiation resistance of TrC1 AKT-E17K cells offer novel perspectives for improving radiosensitivity in cancer cells with aberrant AKT activity by combining IR with inhibitors targeting AKT-dependent regulation of GSH provision.

Motion sickness induced by off-vertical axis rotation (OVAR).

We tested the hypothesis that motion sickness is produced by an integration of the disparity between eye velocity and the yaw-axis orientation vector of velocity storage. Disparity was defined as the magnitude of the cross product between these two vectors. OVAR, which is known to produce motion sickness, generates horizontal eye velocity with a bias level related to velocity storage, as well as cyclic modulations due to re-orientation of the head re gravity. On average, the orientation vector is close to the spatial vertical. Thus, disparity can be related to the bias and tilt angle. Motion sickness sensitivity was defined as a ratio of maximum motion sickness score to the number of revolutions, allowing disparity and motion sickness sensitivity to be correlated. Nine subjects were rotated around axes tilted 10 degrees-30 degrees from the spatial vertical at 30 degrees/s-120 degrees/s. Motion sickness sensitivity increased monotonically with increases in the disparity due to changes in rotational velocity and tilt angle. Maximal motion sickness sensitivity and bias (6.8 degrees/s) occurred when rotating at 60 degrees/s about an axis tilted 30 degrees. Modulations in eye velocity during OVAR were unrelated to motion sickness sensitivity. The data were predicted by a model incorporating an estimate of head velocity from otolith activation, which activated velocity storage, followed by an orientation disparity comparator that activated a motion sickness integrator. These results suggest that the sensory-motor conflict that produces motion sickness involves coding of the spatial vertical by the otolith organs and body tilt receptors and processing of eye velocity through velocity storage.

Frequency-velocity mismatch: a fundamental abnormality in parkinsonian gait.

Gait dysfunction and falling are major sources of disability for patients with advanced Parkinson's disease (PD). It is presently thought that the fundamental defect is an inability to generate normal stride length. Our data suggest, however, that the basic problem in PD gait is an impaired ability to match step frequency to walking velocity. In this study, foot movements of PD and normal subjects were monitored with an OPTOTRAK motion-detection system while they walked on a treadmill at different velocities. PD subjects were also paced with auditory stimuli at different frequencies. PD gait was characterized by step frequencies that were faster and stride lengths that were shorter than those of normal controls. At low walking velocities, PD stepping had a reduced or absent terminal toe lift, which truncated swing phases, producing shortened steps. Auditory pacing was not able to normalize step frequency at these lower velocities. Peak forward toe velocities increased with walking velocity and PD subjects could initiate appropriate foot dynamics during initial phases of the swing. They could not control the foot appropriately in terminal phases, however. Increased treadmill velocity, which matched the natural PD step frequency, generated a second toe lift, normalizing step size. Levodopa increased the bandwidth of step frequencies, but was not as effective as increases in walking velocity in normalizing gait. We postulate that the inability to control step frequency and adjust swing phase dynamics to slower walking velocities are major causes for the gait impairment in PD.

Effect of canal plugging on quadrupedal locomotion in monkey.

The vestibular system plays an important role in controling gait, but where in the labyrinths relevant activity arises is largely unknown. After the semicircular canals are plugged, low frequency (0.01-2 Hz) components of the angular vestibulo-ocular reflex (aVOR) and angular vestibulo-collic reflex (aVCR) are lost, but high frequency (3-20 Hz) components remain. We determined how loss of low frequency canal afference affects limb and head movements during quadrupedal locomotion. Head, body, and limb movements were recorded in three dimensions (3-D) in a cynomolgus monkey with a motion detection system, while the animal walked on a treadmill. All six canals were plugged, reducing the canal time constants from approximately 4.0 sec to approximately 0.07 sec. Major changes in the control of the limbs occurred after surgery. Fore and hind limbs were held farther from the body, producing a broad-based gait. Swing-phase trajectories were inaccurate, and control of medial-lateral limb movement was erratic. These changes in gait were present immediately after surgery, as well as 15 months later, when the animal had essentially recovered. Thus, control of the limbs in the horizontal plane was defective after loss of the low-frequency semicircular canal input and never recovered. Cycle-averaged pitch and roll head rotations, and 3-D head translations were also significantly larger and more erratic after than before surgery. Head rotations in yaw could not be quantified due to intrusion of voluntary head turns. These findings indicate that the semicircular canals provide critical low frequency information to maximize the accuracy of stepping and stabilize the head during normal quadrupedal locomotion.

Effects of the linear vestibulo-ocular reflex on accommodative vergence eye movements.

The aim of the study was to determine whether accommodation to the relative motion of a target along the visual axis of one eye during fore-aft movement of the head could induce accurate vergence over a wide range of viewing distances and frequencies of oscillation, despite lack of vision in the second eye. This was compared to the vergence when both eyes viewed the target. Two rhesus monkeys were trained to fixate a visual target located 216-336 mm in front and along the visual axis of one eye, while being sinusoidally translated in the fore-aft direction. There was no movement of the seeing eye while the other eye converged, regardless of whether there was vision in the converged eye. Gain and phase of the convergence were determined based on the ratio of actual versus expected eye position if the target was accurately fixated. During translation at frequencies from 0.05 to 2 Hz, the eye converged on the target with an eye position gain of approximately 1, and a phase close to zero. When vision was occluded in the converging eye, gains of convergence were 0.6-0.8 Hz up to 2 Hz, and the phases remained close to zero. At low frequencies of fore-aft movement, when the acceleration was negligible, convergence was driven by accommodation in the seeing eye. At higher frequencies, vergence could also be driven by the linear vestibulo-ocular reflex (lVOR). Thus, vision in one nonmoving eye and the lVOR combine to generate convergence over a wide range of frequencies and viewing distances.

Head stabilization by vestibulocollic reflexes during quadrupedal locomotion in monkey.

Little is known about the three-dimensional characteristics of vestibulocollic reflexes during natural locomotion. Here we determined how well head stability is maintained by the angular and linear vestibulocollic reflexes (aVCR, lVCR) during quadrupedal locomotion in rhesus and cynomolgus monkeys. Animals walked on a treadmill at velocities of 0.4-1.25 m/s. Head rotations were represented by Euler angles (Fick convention). The head oscillated in yaw and roll at stride frequencies (approximately 1-2 Hz) and pitched at step frequencies (approximately 2-4 Hz). Head angular accelerations (100-2,500 degrees/s2) were sufficient to have excited the aVOR to stabilize gaze. Pitch and roll head movements were <7 degrees , peak to peak, and the amplitude was unrelated to stride frequency. Yaw movements were larger due to spontaneous voluntary head shifts and were smaller at higher walking velocities. Head translations were small (< or =4 cm). Cynomolgus monkeys positioned their heads more forward in pitch than the rhesus monkeys. None of the animals maintained a forward head fixation point, indicating that the lVCR contributed little to compensatory head movements in these experiments. Significantly, aVCR gains in roll and pitch were close to unity and phases were approximately 180 degrees over the full frequency range of natural walking, which is in contrast to previous findings using anesthesia or passive trunk rotation with body restraint. We conclude that the behavioral state associated with active body motion is necessary to maintain head stability in pitch and roll over the full range of stride/step frequencies encountered during walking.

Relative contribution of walking velocity and stepping frequency to the neural control of locomotion.

Stepping frequency is tightly coupled to walking velocity during natural locomotion. In a recent model, we demonstrated that walking velocity determines stride frequency, governs the active feedback control of the swing and determines the swing phase dynamics that governs foot movement. Here, we questioned whether the swing phase dynamics reflect independent effects of stride frequency and walking velocity. Foot movements were measured with a motion detection system (Optotrak) while subjects walked at 0.6-2.1 m/s on a treadmill. Stepping frequencies of 1.3-2.8 Hz were generated with pacing cues at each walking velocity. In the 'iso-velocity' condition, peak forward toe velocity during the swing phases was related to walking velocity and did not vary with alterations in stride frequency. In the 'iso-frequency' condition, in contrast, stepping frequency altered the relationship between toe acceleration and toe position in the fore-aft direction. The cycle frequency, main sequence (peak velocity vs. amplitude) relationships, and the shape of the phase-plane trajectories of the swing phases also reflected this relationship. The data were modeled by decoupling stepping frequency from walking velocity, while maintaining active feedback control dependent on frequency. The latter predicted both the dominant shape of the phase plane trajectories and the main sequence relationships. Thus, according to the model, walking velocity and stride frequency are independent central variables that control the dynamics of the swing phases and stepping. The ability to decouple stride frequency from walking velocity may help in navigating over uneven terrain or when executing curved trajectories while maintaining a constant velocity.

Rotation axes of the head during positioning, head shaking, and locomotion.

Static head orientations obey Donders' law and are postulated to be rotations constrained by a Fick gimbal. Head oscillations can be voluntary or generated during natural locomotion. Whether the rotation axes of the voluntary oscillations or during locomotion are constrained by the same gimbal is unknown and is the subject of this study. Head orientation was monitored with an Optotrak (Northern Digital). Human subjects viewed visual targets wearing pin-hole goggles to achieve static head positions with the eyes centered in the orbit. Incremental rotation axes were determined for pitch and yaw by computing the velocity vectors during head oscillation and during locomotion at 1.5 m/s on a treadmill. Static head orientation could be described by a generalization of the Fick gimbal by having the axis of the second rotation rotate by a fraction, k, of the angle of the first rotation without a third rotation. We have designated this as a k-gimbal system. Incremental rotation axes for both pitch and yaw oscillations were functions of the pitch but not the yaw head positions. The pivot point for head oscillations was close to the midpoint of the interaural line. During locomotion, however, the pivot point was considerably lower. These findings are well explained by an implementation of the k-gimbal model, which has a rotation axis superimposed on a Fick-gimbal system. This could be realized physiologically by the head interface with the dens and occipital condyles during head oscillation with a contribution of the lower spine to pitch during locomotion.

Three-dimensional kinematics and dynamics of the foot during walking: a model of central control mechanisms.

The foot is a critical interface between the body and supporting surface during walking, but there is no coherent framework on which to model the dynamics of the stance and swing phases. To establish this framework, we studied the rotational and translational dynamics of foot movement in three dimensions with a motion detection system (OPTOTRAK), while subjects walked on a treadmill. Positions, velocities, and durations were normalized to leg-length and gravity. Foot position and rotation at toe-off were closely related to walking velocity. Foot pitch at toe clearance increased with walking velocity, but the medial-lateral and vertical toe positions were unaltered. Phase-plane trajectories along the fore-aft direction, i.e., plots of toe velocity versus position, were circular during the swing phases, with radii proportional to walking velocity. Peak forward, lateral, and upward velocities were linearly related to corresponding excursions, forming main sequences. A second order model predicted the changes in toe position and velocity, and the approximately hyperbolic decrements in duration as a function of walking velocity. The model indicates that the foot is controlled in an overdamped manner during the stance phase and as a feedback-controlled undamped pendulum during the swing. The data and model suggest that the state of the foot at toe-off, set by walking velocity during the stance phase, determines the dynamics of the swing phase. Thus, in addition to determining locomotion kinematics, walking velocity plays a critical role in determining the phase-plane trajectories and main sequence relationships of foot movements during the swing phases.

Dynamics of quadrupedal locomotion of monkeys: implications for central control.

We characterized the three-dimensional kinematics and dynamics of quadrupedal gait of young adult rhesus and cynomolgus monkeys while they walked with diagonal and lateral gaits at 0.4-1.0 m/s on a treadmill. Rigid bodies on the wrist, ankle, and back were monitored by an optical motion detection system (Optotrak). Kinematic data could be normalized using characteristic stride length, reducing variance due to different gait styles, to emphasize common characteristics of swing and stance parameters among animals. Mean swing phase durations fell as walking speed increased, but the swing phase durations increased at each walking velocity as a linear function of increases in amplitude, thereby following a main sequence relationship. The phase plane trajectories of the swing phases, i.e., plots of the relation of the rising and falling limb velocity to limb position in the sagittal (X-Z) plane, had unique dynamic characteristics. Trajectories were separable at each walking velocity and increases in swing amplitude were linearly related to peak swing velocities, thus comprising main sequences. We infer that the swing phase dynamics are set by central neural mechanisms at the onset of the swing phases according to the intended amplitude, which in turn is based on the walking velocity in the stance phases. From the many dynamic similarities between swing phases and rapid eye movements, we further suggest that the swing phases may be generated by neural mechanisms similar to those that produce saccades and quick phases of nystagmus from a signal related to sensed or desired walking velocity.

Robust high-speed binocular 3D eye movement tracking system using a two-radii eye model.

Existing video-based eye movement tracking systems measure three-dimensional eye orientations by assuming the eye is a sphere that rotates around its center at a fixed radius. We found this model inaccurate. We have developed a system that uses a two-radii eye model, which assumes that the eye rotates around two different centers with different radii horizontally and vertically. We found this two-radii model more accurate in estimating the three-dimensional eye positions than the traditional one-radius eye model.

A model-based approach for assessing parkinsonian gait and effects of levodopa and deep brain stimulation.

Gait and balance disturbances are amongst the most disabling features of Parkinson's disease (PD), and are not adequately controlled with currently available medical and surgical therapies. Development of objective quantitative measures of these abnormalities would greatly help in the assessment and the development of therapeutic interventions. Recently, we developed a methodology, using dynamical system theory, for testing gait with a state-of-the-art motion-detection system (OPTOTRAK 3020, Northern Digital, Inc.). We also developed a model of the dynamics of the foot that predicts the stance and swing phase dynamics of normal walking. In the present study, we determined whether model parameters were altered in subjects with PD when they were tested on/off levodopa (LD) and on/off deep brain stimulation (DBS) in a 2 x 2 matrix.

A relational database application in support of integrated neuroscience research.

The development of relational databases has significantly improved the performance of storage, search, and retrieval functions and has made it possible for applications that perform real-time data acquisition and analysis to interact with these types of databases. The purpose of this research was to develop a user interface for interaction between a data acquisition and analysis application and a relational database using the Oracle9i system. The overall system was designed to have an indexing capability that threads into the data acquisition and analysis programs. Tables were designed and relations within the database for indexing the files and information contained within the files were established. The system provides retrieval capabilities over a broad range of media, including analog, event, and video data types. The system's ability to interact with a data capturing program at the time of the experiment to create both multimedia files as well as the meta-data entries in the relational database avoids manual entries in the database and ensures data integrity and completeness for further interaction with the data by analysis applications.

The relation of motion sickness to the spatial-temporal properties of velocity storage.

Tilting the head in roll to or from the upright while rotating at a constant velocity (roll while rotating, RWR) alters the position of the semicircular canals relative to the axis of rotation. This produces vertical and horizontal nystagmus, disorientation, vertigo, and nausea. With recurrent exposure, subjects habituate and can make more head movements before experiencing overpowering motion sickness. We questioned whether promethazine lessened the vertigo or delayed the habituation, whether habituation of the vertigo was related to the central vestibular time constant, i.e., to the time constant of velocity storage, and whether the severity of the motion sickness was related to deviation of the axis of eye velocity from gravity. Sixteen subjects received promethazine and placebo in a double-blind, crossover study in two consecutive 4-day test series 1 month apart, termed series I and II. Horizontal and vertical eye movements were recorded with video-oculography while subjects performed roll head movements of approx. 45 degrees over 2 s to and from the upright position while being rotated at 138 degrees /s around a vertical axis. Motion sickness was scaled from 1 (no sickness) to an endpoint of 20, at which time the subject was too sick to continue or was about to vomit. Habituation was determined by the number of head movements that subjects made before reaching the maximum motion sickness score of 20. Head movements increased steadily in each session with repeated testing, and there was no difference between the number of head movements made by the promethazine and placebo groups. Horizontal and vertical angular vestibulo-ocular reflex (aVOR) time constants declined in each test, with the declines being closely correlated to the increase in the number of head movements. The strength of vertiginous sensation was associated with the amount of deviation of the axis of eye velocity from gravity; the larger the deviation of the eye velocity axis from gravity, the more severe the motion sickness. Thus, promethazine neither reduced the nausea associated with RWR, nor retarded or hastened habituation. The inverse relationship between the aVOR time constants and number of head movements to motion sickness, and the association of the severity of motion sickness with the extent, strength, and time of deviation of eye velocity from gravity supports the postulate that the spatiotemporal properties of velocity storage, which are processed between the nodulus and uvula of the vestibulocerebellum and the vestibular nuclei, are likely to represent the source of the conflict responsible for producing motion sickness.

Compensatory and orienting eye movements induced by off-vertical axis rotation (OVAR) in monkeys.

Nystagmus induced by off-vertical axis rotation (OVAR) about a head yaw axis is composed of a yaw bias velocity and modulations in eye position and velocity as the head changes orientation relative to gravity. The bias velocity is dependent on the tilt of the rotational axis relative to gravity and angular head velocity. For axis tilts <15 degrees, bias velocities increased monotonically with increases in the magnitude of the projected gravity vector onto the horizontal plane of the head. For tilts of 15-90 degrees, bias velocity was independent of tilt angle, increasing linearly as a function of head velocity with gains of 0.7-0.8, up to the saturation level of velocity storage. Asymmetries in OVAR bias velocity and asymmetries in the dominant time constant of the angular vestibuloocular reflex (aVOR) covaried and both were reduced by administration of baclofen, a GABA(B) agonist. Modulations in pitch and roll eye positions were in phase with nose-down and side-down head positions, respectively. Changes in roll eye position were produced mainly by slow movements, whereas vertical eye position changes were characterized by slow eye movements and saccades. Oscillations in vertical and roll eye velocities led their respective position changes by approximately 90 degrees, close to an ideal differentiation, suggesting that these modulations were due to activation of the orienting component of the linear vestibuloocular reflex (lVOR). The beating field of the horizontal nystagmus shifted the eyes 6.3 degrees /g toward gravity in side down position, similar to the deviations observed during static roll tilt (7.0 degrees /g). This demonstrates that the eyes also orient to gravity in yaw. Phases of horizontal eye velocity clustered ~180 degrees relative to the modulation in beating field and were not simply differentiations of changes in eye position. Contributions of orientating and compensatory components of the lVOR to the modulation of eye position and velocity were modeled using three components: a novel direct otolith-oculomotor orientation, orientation-based velocity modulation, and changes in velocity storage time constants with head position re gravity. Time constants were obtained from optokinetic after-nystagmus, a direct representation of velocity storage. When the orienting lVOR was combined with models of the compensatory lVOR and velocity estimator from sequential otolith activation to generate the bias component, the model accurately predicted eye position and velocity in three dimensions. These data support the postulates that OVAR generates compensatory eye velocity through activation of velocity storage and that oscillatory components arise predominantly through lVOR orientation mechanisms.

Spatial orientation of caloric nystagmus.

The spatial orientation of the slow-phase eye velocity of caloric nystagmus was investigated in cynomolgus monkeys after all six semicircular canals had been plugged. Normal animals generate responses that have dominant convective components produced by movement of the endolymph in the lateral canal toward or away from gravity. As a result, the direction of horizontal slow-phase velocity induced by cold-water irrigation changes direction with changes in head position with regard to gravity. Plugging produced a dense overgrowth of bone that blocked the flow of endolymph, but the end organs were intact. Robust caloric nystagmus was elicited after recovery, but the horizontal (yaw) component was now always toward the stimulated (ipsilateral) side, regardless of head position re gravity. The induced caloric nystagmus had strong spatial orientation properties after canal plugging. With animals upright, the three-dimensional velocity vector of the caloric nystagmus was close to the yaw axis with small vertical and roll components. Roll components became stronger in supine and prone positions and vertical components were enhanced in the right- and left-side down positions. In each instance, the addition of the roll and vertical components moved the velocity vector of the nystagmus closer to the spatial vertical. Modeling supported the postulate that the caloric nystagmus after canal plugging is influenced by three factors: (1) a reduction in neural activity in the ampullary nerves on the stimulated side due to cooling of the nerves; (2) contraction of the endolymph in the closed space between the cupula and the plug due to cooling, which resulted in deflection of the cupula and hair cells toward the plug (ampullofugal deflection); and (3) alignment of eye velocity to gravity due to the orientation properties of velocity storage. Although convection is the most prominent factor in producing caloric responses in the normal state, our results suggest that alteration of nerve activity due to thermal effects, endolymph contraction or expansion, and velocity storage are also likely to contribute to the total response.