Striatal dysregulation of Cdk5 alters locomotor responses to cocaine, motor learning, and dendritic morphology.

Motor learning and neuro-adaptations to drugs of abuse rely upon neuronal signaling in the striatum. Cyclin-dependent kinase 5 (Cdk5) regulates striatal dopamine neurotransmission and behavioral responses to cocaine. Although the role for Cdk5 in neurodegeneration in the cortex and hippocampus and in hippocampal-dependent learning has been demonstrated, its dysregulation in the striatum has not been examined. Here we show that strong activation of striatal NMDA receptors produced p25, the truncated form of the Cdk5 co-activator p35. Furthermore, inducible overexpression of p25 in the striatum prevented locomotor sensitization to cocaine and attenuated motor coordination and learning. This corresponded with reduced dendritic spine density, increased neuro-inflammation, altered dopamine signaling, and shifted Cdk5 specificity with regard to physiological and aberrant substrates, but no apparent loss of striatal neurons. Thus, dysregulation of Cdk5 dramatically affects striatal-dependent brain function and may be relevant to non-neurodegenerative disorders involving dopamine neurotransmission.

Antivascular effects of combretastatin A4 phosphate in breast cancer xenograft assessed using dynamic bioluminescence imaging and confirmed by MRI.

Bioluminescence imaging (BLI) has found significant use in evaluating long-term cancer therapy in small animals. We have now tested the feasibility of using BLI to assess acute effects of the vascular disrupting agent combretastatin A4 phosphate (CA4P) on luciferase-expressing MDA-MB-231 human breast tumor cells growing as xenografts in mice. Following administration of luciferin substrate, there is a rapid increase in light emission reaching a maximum after about 6 min, which gradually decreases over the following 20 min. The kinetics of light emission are highly reproducible; however, following i.p. administration of CA4P (120 mg/kg), the detected light emission was decreased between 50 and 90%, and time to maximum was significantly delayed. Twenty-four hours later, there was some recovery of light emission following further administration of luciferin substrate. Comparison with dynamic contrast-enhanced MRI based on the paramagnetic contrast agent Omniscan showed comparable changes in the tumors consistent with the previous literature. Histology also confirmed shutdown of tumor vascular perfusion. We believe this finding provides an important novel application for BLI that could have widespread application in screening novel therapeutics expected to cause acute vascular changes in tumors.

Iterative reconstruction method for light emitting sources based on the diffusion equation.

Bioluminescent imaging (BLI) of luciferase-expressing cells in live small animals is a powerful technique for investigating tumor growth, metastasis, and specific biological molecular events. Three-dimensional imaging would greatly enhance applications in biomedicine since light emitting cell populations could be unambiguously associated with specific organs or tissues. Any imaging approach must account for the main optical properties of biological tissue because light emission from a distribution of sources at depth is strongly attenuated due to optical absorption and scattering in tissue. Our image reconstruction method for interior sources is based on the deblurring expectation maximization method and takes into account both of these effects. To determine the boundary of the object we use the standard iterative algorithm-maximum likelihood reconstruction method with an external source of diffuse light. Depth-dependent corrections were included in the reconstruction procedure to obtain a quantitative measure of light intensity by using the diffusion equation for light transport in semi-infinite turbid media with extrapolated boundary conditions.

Robust controller for tremor suppression at musculoskeletal level in human wrist.

Tremor is a rhythmical and involuntary oscillatory movement of a body part and it is one of the most common movement disorders. Orthotic devices have been under investigation as a noninvasive tremor suppression alternative to medication or surgery. The challenge in musculoskeletal tremor suppression is estimating and attenuating the tremor motion without impeding the patient's intentional motion. In this research a robust tremor suppression algorithm was derived for patients with pathological tremor in the upper limbs. First the motion in the tremor frequency range is estimated using a high-pass filter. Then, by applying the backstepping method the appropriate amount of torque is calculated to drive the output of the estimator toward zero. This is equivalent to an estimation of the tremor torque. It is shown that the arm/orthotic device control system is stable and the algorithm is robust despite inherent uncertainties in the open-loop human arm joint model. A human arm joint simulator, capable of emulating tremorous motion of a human arm joint was used to evaluate the proposed suppression algorithm experimentally for two types of tremor, Parkinson and essential. Experimental results show 30-42 dB (97.5-99.2%) suppression of tremor with minimal effect on the intentional motion.

A Multi-Camera System for Bioluminescence Tomography in Preclinical Oncology Research.

Bioluminescent imaging (BLI) of cells expressing luciferase is a valuable noninvasive technique for investigating molecular events and tumor dynamics in the living animal. Current usage is often limited to planar imaging, but tomographic imaging can enhance the usefulness of this technique in quantitative biomedical studies by allowing accurate determination of tumor size and attribution of the emitted light to a specific organ or tissue. Bioluminescence tomography based on a single camera with source rotation or mirrors to provide additional views has previously been reported. We report here in vivo studies using a novel approach with multiple rotating cameras that, when combined with image reconstruction software, provides the desired representation of point source metastases and other small lesions. Comparison with MRI validated the ability to detect lung tumor colonization in mouse lung.

The role of Cdk5 in neuroendocrine thyroid cancer.

Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer that originates from calcitonin-secreting parafollicular cells, or C cells. We found that Cdk5 and its cofactors p35 and p25 are highly expressed in human MTC and that Cdk5 activity promotes MTC proliferation. A conditional MTC mouse model was generated and corroborated the role of aberrant Cdk5 activation in MTC. C cell-specific overexpression of p25 caused rapid C cell hyperplasia leading to lethal MTC, which was arrested by repressing p25 overexpression. A comparative phosphoproteomic screen between proliferating and arrested MTC identified the retinoblastoma protein (Rb) as a crucial Cdk5 downstream target. Prevention of Rb phosphorylation at Ser807/Ser811 attenuated MTC proliferation. These findings implicate Cdk5 signaling via Rb as critical to MTC tumorigenesis and progression.

High-throughput quantitative bioluminescence imaging for assessing tumor burden.

Bioluminescence imaging (BLI) has emerged during the past 5 years as the preeminent method for rapid, cheap, facile screening of tumor growth and spread in mice. Both subcutaneous and orthotopic tumor models are readily observed with high sensitivity and reproducibility. User-friendly commercial instruments exist and, increasingly, luciferase-expressing tumor cells are available in academic institutions or commercially. There is an increasing literature on routine use of BLI for assessing chemotherapeutic efficacy, drug combinations, dosing, and timing. In addition, BLI may be applied to more sophisticated questions of molecular biology by including specific promoter sequences. This chapter will describe routine methods used to support multiple investigators in our small animal imaging resource.

The use of histone deacetylase inhibitor FK228 and DNA hypomethylation agent 5-azacytidine in human bladder cancer therapy.

The long-term disease-free survival in patients with metastatic transitional cell carcinoma (TCC) is still considerably low. Novel chemotherapeutic agents are needed to decrease the morbidity and mortality of TCC. In this study, we have evaluated several epigenetic modifiers for their therapeutic application in bladder cancer. Both histone deacetylase inhibitors (FK228, TSA) and DNA hypomethylating agent (5-Azacytidine) were tested using in vitro assays such as cell viability, cell cycle analysis and western blot to determine their mechanisms of action. Drug combination experiments were also designed to study any additive or synergistic effects of these agents. In addition, two bladder cancer xenograft models (one subcutaneous and one orthotopic) were employed to assess the therapeutic efficacy of these agents in vivo. Three agents exhibited various growth inhibitory effects on 5 different TCC cell lines in a dose- and time-dependent manner. In addition to G2/M cell cycle arrest, FK228 is more potent in inducting apoptosis than the two other single agents, and combination of both FK228 and 5-Aza further enhances this effect. p21 induction is closely associated with FK228 or TSA but not 5-Aza, which is mediated via p53-independent pathway. Consistent with in vitro results, FK228 exhibited a significant in vivo growth inhibition of TCC tumor in both subcutaneous and orthotopic xenograft models. FK228 is a potent chemotherapeutic agent for TCC in vivo with minimal undesirable side effects. The elevated p21 level mediated via p53 independent pathway is a hallmark of FK228 mechanism of action.

Reduction in normalized bone elasticity following long-term bisphosphonate treatment as measured by ultrasound critical angle reflectometry.

Using an improved version of ultrasound critical angle reflectometry, the bone quality of cortical and trabecular bone was assessed in vivo by measuring elastic moduli (normalized for bone density) at both principal axes, referred to as the minimum and maximum normalized elasticities. The measurements were made in 30 normal premenopausal women, 30 normal postmenopausal women, 22 untreated postmenopausal women with osteoporosis, 74 postmenopausal women with osteoporosis or osteopenia on bisphosphonate treatment, and 32 patients with renal transplantation (16 women and 16 men) taking steroids. Cortical elasticity was higher than trabecular elasticity; both declined slightly and non-significantly with age in normal women. Among untreated postmenopausal women with osteoporosis, cortical maximum normalized elasticity (E(cmax)) remained within 95% prediction intervals of normal women. Among patients on bisphosphonate, E(cmax) was low in the majority of patients. E(cmax) was significantly more depressed among those taking the drug > or =3 years than <3 years (22.1% below normal premenopausal women versus 17.2%, P =0.001), and among those with incident non-spinal fractures than without (75.9 vs. 81.5%, P =0.008). E(cmax) was independent of bone mineral density at the calcaneus. Most patients with renal transplantation had low E(cmax), with a mean 20.8% below the normal premenopausal mean. Qualitatively similar findings were found with cortical minimum elasticity and with trabecular minimum and maximum elasticities. Thus, the material bone quality of cortical and trabecular bone may be impaired following bisphosphonate treatment, as in renal transplantation on steroids.

Validating bioluminescence imaging as a high-throughput, quantitative modality for assessing tumor burden.

Bioluminescence imaging (BLI) is a highly sensitive tool for visualizing tumors, neoplastic development, metastatic spread, and response to therapy. Although BLI has engendered much excitement due to its apparent simplicity and ease of implementation, few rigorous studies have been presented to validate the measurements. Here, we characterize the nature of bioluminescence output from mice bearing subcutaneous luciferase-expressing tumors over a 4-week period. Following intraperitoneal or direct intratumoral administration of luciferin substrate, there was a highly dynamic kinetic profile of light emission. Although bioluminescence was subject to variability, strong correlations (r >.8, p <.001) between caliper measured tumor volumes and peak light signal, area under light signal curve and light emission at specific time points were determined. Moreover, the profile of tumor growth, as monitored with bioluminescence, closely resembled that for caliper measurements. The study shows that despite the dynamic and variable nature of bioluminescence, where appropriate experimental precautions are taken, single time point BLI may be useful for noninvasive, high-throughput, quantitative assessment of tumor burden.