Retreat 2004 M.D./Ph.D. student abstracts
Cerebral and Cerebellar Activation during Arithmetic Calculation and Timed Motor Tasks in Men with Fragile X-Associated Tremor/Ataxia Syndrome
PSTP Student: Teresa Barcellos
Mentor: Dr. Susan Rivera
Collaborators: David N, and Henry M
Fragile X syndrome, the most common inherited cause of mental retardation, has long been known to result from the expansion of a trinucleotide (CGG) repeat in the FMR1 gene on the X chromosome from fewer than 50 to more than 200 repeats. An expansion with a repeat length between 50 and 200 is termed a premutation and is highly susceptible to further expansion in female gametogenesis. Historically, carriers of the fragile X premutation were believed to be phenotypically normal; however, recent research has demonstrated the presence of a neurodegenerative disorder, termed Fragile X-associated tremor/ataxia syndrome, or FXTAS, in older male carriers. FXTAS is a progressive disorder characterized by worsening intention tremor, gait ataxias, parkonsonian-type rigidity, and eventual cognitive decline. Structural MRI studies of FXTAS patients have shown generalized cerebral and cerebellar atrophy as well as white matter hyperintensity of the middle cerebellar peduncle (MCP), while post-mortem neuropathological analysis showed cerebral neuronal and astrocytic intranuclear inclusions and cerebellar Purkinje cell axonal degradation. In our study, we attempt to characterize functional neuropathologies in these patients using functional magnetic resonance imaging (fMRI) during an arithmetic calculation task and a motor timing task. Although similar fMRI studies have been used in the past to demonstrate differing activation patterns in fragile X patients and their normal counterparts, to date no such study has been carried out using the FXTAS population. Since the structural and clinical evidence suggests that the changes observed in FXTAS are largely due to changes in the MCP and cerebellum, we expected that carriers would show a change in activation patterns, specifically an increase in cerebellar activity during the motor timing task and decreased recruitment of the cerebellum when performing calculations, similar to that seen in patients with other cerebellar disorders, such as intention tremor and spinocerebellar ataxias. Thus far, our results have been consistent with this hypothesis. During the arithmetic calculation task, FXTAS subjects show little, if any, activation of the cerebellum, while controls show robust cerebellar recruitment. Conversely, FXTAS patients show significantly more cerebellar activation than controls during the motor timing task. Cerebellar activation also correlates positively with FXTAS severity in the motor task and negatively in the calculation task. Additionally, the FXTAS and control patients show differing patterns of cerebral activation.
Potential Role of Candidate plasticity-related gene 2 (cpg2) in Excitatory Synapse Formation: A Preliminary Study
PSTP Student: Jolene M. Chang
Mentor: A. Kimberley McAllister
Collaborators: Cottrell J, and Nedivi E
Candidate plasticity related gene 2 (cpg2) is a gene that is upregulated in response to activity in the rat dentate gyrus. Expressed only in the brain and in areas of the brain that are capable of synaptic plasticity, cpg2 acts as a postsynaptic negative regulator of glutamate receptor endocytosis. Because of its role in glutamate receptor endocytosis, my research this summer focused on studying cpg2 and synapse formation in young developing neurons in vitro. Imaging cpg2-labeled neurons with a laser-scanning confocal microscope, cpg2 staining at 4 days in vitro is diffuse across dendrites. However, at day 7 and day 10, the diffuse cpg2 staining becomes progressively more punctate at synapses and also colocalizes mostly with NMDA receptors. Thus, cpg2 is located at the right time and place in order to be regulating excitatory synaptic formation in young developing in vitro neurons. Future experiments will confirm cpg2’s functional role in synapse formation in neuronal development. In developing synapses, glutamate receptor trafficking to its target destination at synapses in neuronal dendrites is an important step in excitatory synapse formation. In this way, cpg2 can be an important factor in excitatory synapse formation by regulating glutamate receptor insertion.
KLF8 and IL-8-mediated Androgen-Independent Growth in LNCaP
PSTP Student: Yu-Ming Chang
Mentors: Drs. Hsing-Jien Kung, Dr. Christopher P. Evans
Prostate cancer (CaP) is the most common cancer in American men and its contribution to cancer-related deaths in this population is second only to lung cancer. Although anti-androgen therapy results in cancer cell apoptosis during its growth both in vivo and in vitro, CaP eventually develops androgen independence and proliferates uninhibited despite treatment, thus leading to patient morbidity and mortality.
Interleukin-8 is a CXC chemokine initially identified as a regulator for the recruitment and trafficking of leukocytes to sites of inflammation. When stimulated with exogenous IL-8, LNCaP, an androgen-dependent prostate cancer cell line has been shown to become AI and to phosphorylate FAK (focal adhesion kinase), a non-receptor tyrosine kinase. The role of FAK in IL-8-induced AI of LNCaP however, remains unclear. Krüppel-like Factor 8 (KLF8) was initially identified as a CACCC-box binding protein that associates with CtBP and shown to be a transcriptional repressor. Subsequently, KLF8 was found to be a downstream target of FAK in its regulation of Cyclin D1 expression levels.
I am thus interested in determining if IL-8-facilitated AI growth of LNCaP is in part mediated by FAK and KLF8. In my experiments, LNCaP cells were placed in charcoal-dextrin-stripped (CS) androgen-minimal media for four days. This was followed by the addition of 10 µM of exogenous recombinant hIL-8. The resulting RNA and protein from these cells were harvested at various post-treatment time points for further analysis by RT-PCR and Western Blot respectively. Since IL-8 treatment had been shown to recruit the androgen receptor (AR) to the promoter of prostatic specific antigen. I also examined whether KLF8 plays a role in this process through the co-immunoprecipitation of the AR followed by probing of KLF8. Furthermore, I examined whether the presence of androgen in cell culture media has an effect on the expression of KLF8.
RT-PCR of IL-8 treated LNCaP cells revealed an up-regulation of KLF8 RNA at about 16 hours post-treatment. Similarly, Western Blot analysis showed that KLF8 protein level is increased at about 24 hours post IL-8 treatment. Co-immunoprecipitation experiments, however, failed to demonstrate an association between the androgen receptor and KLF8. Lastly, Western Blot analysis of LNCaP grown in culture media with androgen presence revealed an additional band slightly heavier than that of the KLF8 band from LNCaP treated with CS media. I therefore conclude that KLF8 may play a role in IL-8 induced AI growth of LNCaP, though not through its interaction with the AR. Additionally, the presence of androgen in culture media may regulate KLF8 in a post-translational manner.
A COMPUTER-ASSISTED INTRAVITAL MICROSCOPY STUDY ON MICROVASCULAR CHANGES IN HEMORRHAGIC SHOCK AND RESUSCITATION IN A CANINE HYPOVOLEMIA MODEL
PSTP Student: Patricia L. Duong
Mentor: Dr. Anthony T. Cheung
Microvascular changes in moderate hypovolemia and resuscitation treatment using blood substitutes [6% hetastarch and HB-200 (a hemoglobin-based oxygen carrier)] with autologous (shed) blood as control were studied. In addition, systemic function and oxygenation changes were monitored continuously for correlation. Nine animals were studied -- after splenectomy, each animal was hemorrhaged (MAP reduced to 50mm Hg; ~40% blood loss=32-36mL/kg) and randomly assigned to 3 resuscitation groups: hetastarch (n=3), HB-200 (n=3), and control (n=3). Computer-assisted intravital microscopy (CAIM) was used to videotape and objectively quantify microvascular changes in the conjunctival microcirculation during prehemorrhagic, posthemorrhagic, and resuscitation phases of the study. Prehemorrhagic microvascular characteristics were similar in all animals (diameter of venules=41±12µm, A:V ratio=~1:2, red-cell velocity=0.5±0.3mm/s). All animals showed similar significant (P<0.01) posthemorrhagic changes: ~19% decrease in vessel diameter (diameter of venules=34±7µm), A:V ratio=extremely variable, and ~80% increase in red-cell velocity (0.9±0.5mm/s). Control blood resuscitation restored microvascular changes to prehemorrhagic baseline values (diameter=39±6µm, A:V ratio=~1:2, red-cell velocity=0.6±0.4mm/s). Hetastarch (non-oxygen carrier) and HB-200 (oxygen carrier) also restored microvascular changes to prehemorrhagic values in similar manner (Hetastarch diameter=38±7µm, A:V ratio=~1:2, red-cell velocity=0.5±0.4mm/s; HB-200 diameter=38±3µm, A:V ratio=~1:2, red-cell velocity=0.6±0.4mm/s) despite differences in oxygen-carrying capability. The results indicate that either volume replenishment and not oxygen-carrying capability is needed for treatment of moderate hypovolemia or that standard OR methods cannot detect oxygenation changes at the tissue level.
X-Band Photoinjector/High Gradient Accelerator Based Light Source
PSTP Student: William J. Frederick
Mentor: Dr. John Heritage
Collaborators: Vlieks AE, DeStefano C, Caryotakis G, Luhmann NC, Martin D, and Landahl E
A compact source of monochromatic ultrashort X-ray pulses tunable in the 10 to 85 keV range is obtained from Compton scattering of intense sub-picosecond optical pulses with relativistic electron bunches. Unlike conventional large national laboratory scale synchrotron radiation sources, the relativistic Compton device permits compact vacuum components including a single X-band Klystron, a 5.5 cell photoinjector, and a 1.05-meter, high-gradient linear accelerator. Optical radiation is generated by a tabletop synchronized Titanium:Sapphire femtosecond laser oscillator and amplifier system. Finding Compton x-rays produced on sub-picosecond timescales have addressed unique timing and detection challenges. Our source holds great potential as a probe of inner shell bound electrons in heavy atoms for medical diagnostics and theraputics, as well as for studies of fundamental atomic-scale physics. In particular, X-ray phototherapy experiments using existing (synchrotron) monoenergetic x-ray sources as proof of principle demonstrations show promise for the utility of this device.
Amiloride Kills Malignant Glioma Cells Independent of Its Inhibition of the Sodium-Hydrogen Exchanger.
PSTP Student: Manu Hegde
Mentor: Fred Gorin, M.D., Ph.D.
Collaborators: Harley B, Roscoe J, and Cala P
Previously, we demonstrated that malignant glioma cell lines have increased intracellular pH (pHi) as a result of increased activities of the type I sodium/hydrogen exchanger (NHE1). This alkalotic pHi of 7.2 to 7.4 is favorable for augmented glycolysis, DNA synthesis, and cell cycle progression. Conversely, reductions in pHi have been associated with reduced rates of proliferation in transformed cell types. The effects of reducing pHi directly and by NHE1 inhibition on human malignant glioma cells were systematically compared with those on primary rat astrocytes. Neither cariporide, nor direct acidification to pHi 6.9 altered the proliferative rates or viabilities of human U87 or U118 malignant glioma cell lines. However, amiloride significantly impaired glioma cell proliferation and viability while not affecting astrocytes at concentrations (500 µM) that exceeded its inhibition of NHE1 in glioma cells (IC50 17 µM). Preventing a reduction of pHi did not alter the drug’s antiproliferative and cytotoxic effects on glioma cells. These findings indicated that amiloride’s cytotoxic effects on glioma cells are independent of its ability to inhibit NHE1 or to reduce intracellular pHi. The amiloride derivative 2,4 dichlorobenzamil (DCB) inhibits the sodium-calcium exchanger (NCX) and was both antiproliferative and cytotoxic to glioma cells at low doses (20 µM). By contrast, KB-R7943 [(2-[2-[4-nitrobenzyloxy]phenyl]ethyl)-isothioureamethanesulfonate] preferentially blocks sodium-dependent calcium influx by NCX (reverse mode) and was nontoxic to glioma cells. It is proposed that DCB (20 µM) and amiloride (500 µM) impair calcium efflux by NCX, leading to elevations of intracellular calcium that initiate a morphologically necrotic, predominantly caspase-independent glioma cell death.
Three Surgical Approaches for Lumbar Corpectomy Using Allograft and Synthetic Devices: An in-vivo Calf Model to Assess Spinal Fusion
PSTP Student: Philbert Y. Huang
Mentor: Dr. Nesrin Sarigul-Klijn
Fractures, tumors, bacterial infections and other diseases affecting vertebral bodies can result in structural degradation leading to spinal instability and/or neural impingement. For many of these conditions it is mandatory to reconstruct the destabilized spine following damaged vertebral body resection, or vertebral corpectomy. Anterior bone grafting is often performed. However, bone grafting alone does not provide spinal stability immediately after the surgery. Therefore, either anterior or posterior spinal instrumentation in the form of a plate or rods, combined with anterior bone graft, has been advocated for the restoration of vertebral column stability. This stability is achieved by a process known as spinal fusion, during which the graft device/plate construct provides an environment where a bony bridge can form across the graft device. To further improve patient care, it is critical to evaluate these instruments through biomechanical testing. Since it is important to perform evaluations in the context of the healing abilities of soft tissue and bone we are developing an in vivo calf model for analyzing the physiological interactions between the bone and implant. Lumbar spinal instability will be created in the animal model by a corpectomy procedure at a given level. Next, the following vertebral body replacement methods will be implemented in the animal model: 1) tibial/metatarsal allograft; 2) Synthes Synmesh Spacer; and 3) Synthes Synex Device.
These strut implants will be used with a Synthes Thoracolumbar Spine Locking Plate. Following an appropriate postoperative period, the animals will be sacrificed and their lumbar spines harvested. This will allow for radiological, biomechanical, and histological assessment. Radiological assessment is that which is traditionally used to evaluate fusion in humans. Biomechanical testing parameters will include subjecting the segment to flexion/extension, lateral bending, and axial rotation. Load versus deformation data will allow for the determination of torsional, axial, and flexural stiffnesses. Another consideration will be the use of strain gauges attached to the surfaces of the implants to provide direct measurement of axial and flexural strains. Stiffness and/or strain data can then be compared across the fused vertebral constructs to determine if one is superior to the others. Through histological assessment we may characterize the extent of trabecular or mature bone growth, especially at the implant-bone interface. These studies should provide valuable insight for the design and development of the next generation of implants for spinal stabilization.
SMRT and N-CoR corepressors are regulated by distinct kinase signaling pathways
PSTP Student: Brian Jonas
Mentor: Dr. Martin Privalsky
Nuclear Hormone Receptor (N-CoR) and Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptors (SMRT) are corepressor paralogs that partner with, and mediate transcriptional repression by a wide variety of metazoan transcription factors, including nuclear hormone receptors. Although encoded by distinct genetic loci, N-CoR and SMRT share substantial sequence interrelatedness, form analogous assemblies with histone deacetylases and auxiliary factors, can interact with overlapping sets of transcription factor partners, and exert overlapping functions in cells. SMRT is subject to negative regulation by MAP kinase signaling pathways operating downstream of growth factor and stress-signaling pathways. We report here that whereas activation of MEKK1 leads to phosphorylation of SMRT, its dissociation from its transcription factor partners in vivo and in vitro, and its redistribution from the cell nucleus to a cytoplasmic compartment, N-CoR is refractory to all these forms of regulation. In contrast to this MAP kinase cascade, other signal transduction pathways operating downstream of growth factor/cytokine receptors appear able to affect both corepressor paralogs. Our results indicate that SMRT and N-CoR are embedded in distinct regulatory networks, and that two corepressors interpret growth factor, cytokine, differentiation, and pro-survival signals differently.
LONG LASTING ADAPTATION IMPROVES RESOLUTION OF ROD PHOTORECEPTORS
PSTP Student: Claudia M. Krispel
Mentor: Dr. Marie E. Burns
Rod photoreceptors adapt to light by speeding the incremental response kinetics and decreasing sensitivity, both of which help to extend the range of light intensities over which rods can respond. We recently reported a form of adaptation that speeds response recovery for many tens of seconds after the adapting stimulus has been extinguished. The long-lasting nature of this adaptation is unusual since most other (i.e. “classical”) forms of light adaptation require the presence of steady light. In this study, we aimed to determine the relationship between this long-lasting form of adaptation and classical adaptation, and to begin to examine how long-lasting adaptation alters rod function.
Using suction electrodes, we recorded the responses of mouse rods to flashes of light either in the presence or absence of steady dim light that suppressed the circulating current by up to 30% (to induce classical adaptation). Long-lasting adaptation was induced by exposing the rods to several minutes of just-saturating light. We found that long-lasting adaptation speeded response recovery to a similar extent and faded with a similar slow time course despite the presence of dim steady light. These results show that classical adaptation to dim light does not interfere with induction, expression, or extinction of long lasting adaptation, suggesting that long lasting adaptation is probably mediated by an independent mechanism. To test whether long- lasting adaptation improves the temporal responsiveness of the rod, we measured the rod’s ability to follow a flickering stimulus before and after induction of long lasting adaptation. We found long lasting adaptation improved the ability of cells to signal the frequency of a flickering stimulus. Our experiments suggest that long lasting adaptation is mediated by a novel adaptation mechanism and may help to improve the temporal resolution of the rod system across the mesopic range of light intensities.
Elimination of Synaptic Complexes by Sema3F Signaling Initiates Stereotyped Axon Pruning in the Hippocampus
PSTP Student: Lawrence K. Low
Mentor: Hwai-Jong Cheng
Collaborators: Liu XB, and Jones EG
Sema3F signaling is required for stereotyped pruning of long axon collaterals in the vertebrate CNS; however, a cellular basis for Sema3F on stereotyped pruning has not been determined. In the immature mouse hippocampus, we found that mossy fiber axon collaterals form transient synaptic complexes with basal dendrites of CA3 pyramidal cells. When Sema3F signaling is present, these synaptic complexes stop maturing and are removed prior to stereotyped pruning by axon retraction. When Sema3F signaling is blocked through loss of function mutations for its receptors, Plexin-A3 or Neuropilin-2, the synaptic complexes continue to mature and, as a result, the collaterals are not pruned. Thus, our results suggest a new role for Semaphorin signaling in the vertebrate CNS—that intact Sema3F signaling initiates stereotyped pruning of long axon collaterals by eliminating transient synaptic complexes.
Shiloh M. Martin (and David L. Kukis)
Laboratory of Dr. Joseph Tuscano in collaboration with the laboratory of Dr. Julie Sutcliffe-Goulden.
ABSTRACT for PSTP retreat Sept 17, 2004
B lymphocyte signaling via surface molecule CD22 has been shown to increase proliferation in vitro. HB22.7 is a mouse monoclonal antibody that blocks CD22, resulting in apoptosis in neoplastic B cells. Previous studies in the lab have shown that HB22.7 reduces tumor volume in mice with Raji (a non-Hodgkin’s lymphoma cell line) tumors. Currently we are developing a 64Cu-DOTA-HB22.7 antibody. DOTA is a macrocyclic chelator that links the radionuclide to the antibody. 64Cu has positron emissions that will allow us not only to visualize tumor targeting of the antibody but also to track tumor regression over time in vivo via a microPET scanner. Additionally, 64Cu has low energy B particle emissions that can be therapeutic. As of this writing, DOTA has been successfully conjugated to HB22.7 in varying ratios and all conjugated antibodies were determined to maintain immunogenicity. Before mouse studies begin, a serum stability study is planned to assure that 64Cu will remain bound to HB22.7 and not bind to various serum proteins. It is hoped that 64Cu-DOTA-HB22.7 could become a real possibility for future treatment of Non-Hodgkin’s lymphoma.
Noah Merin PSTP Student V
Autism is a biological disorder that pervasively affects development. There is a need to identify children at risk for autism as early as possible, and to develop optimal treatments. Evidence is strong that symptoms of autism are discernable in the earliest ages thus far examined, 8 to 12 months. Since the pathology of autism most likely has its beginnings in prenatal development, there is reason to believe that signs of risk may be present quite early. This project seeks to identify behavioral markers of autism in early infancy.
Method: Three- and six-month old infants who have an older sibling with autism, and who therefore have an elevated risk of developing the disorder, will be studied in a battery of social processing tasks. We will use eye tracking methodology to examine infant gaze patterns during live reciprocal interaction with the mother. Other experiments will investigate face expression processing, and object knowledge. Performance on these tasks will be compared to two groups of control infants: typically developing infants, and infants at risk for developmental delay. Subjects will participate in additional experimental testing, developmental assessment, and be followed for diagnosis, until 42 months of age. At the conclusion of the study, we will compare the performance of children who were diagnosed with autism to control subjects, to see if behavioral differences were apparent at the early developmental timepoints.
Molecular Cloning and Functional Identification of MERG1a Potassium Channel in Mouse Cochlea.
PSTP Student: Karen Mu
Mentor: Dr. Ebenezer Yamoah
Collaborators: Nie L, Feng W, Gratton MA, and Vazquez AE
Alterations in K+ homeostasis in the cochlear duct have profound effects on the transduction of auditory signals. Previous pharmacological, biochemical, and functional studies have identified candidate ion transporters, pumps, and channels that are involved in the K+ regulation in the cochlea. The present work is the first to demonstrate the molecular identification and cellular localization of ERG1a channel in mouse inner ear (MERG1a). A cDNA fragment containing the complete open reading frame of merg1a was amplified from cochlea by RT-PCR and cloned into pCRII-TOPO. The amino acid sequence of the putative protein shows typical characteristics of erg K+ channels with six transmembrane domains (S1-S6) and the pore region. The sequence comparison showed that mouse inner ear-specific MERG1a shares high homology with ERG1a proteins from other tissue types and species.
The cellular distribution of MERG1a in the cochlea was investigated using both immunoperoxidase and immunofluorescence techniques. The highest levels of immuno-reactivity were found in the intermediate cells of stria vascularis and the spiral ganglion cells. In addition, low levels of expression were detected in hair cells, type II and V fibrocytes of the spiral ligament and fibrocytes underlying the interdental cells of the spiral limbus. To characterize the functional and pharmacological phenotype of the channel, the channel was expressed in a heterologous expression system (Xenopus oocytes). In Xenopus oocytes, MERG1a channel is K+ permeable, E4031- and rBeKm-1-sensitive, activates at relatively negative potentials, and shows characteristic rapid inactivation reflected as inward rectification at depolarized potentials: a typical property of ERG K+ channel family.
Title: One-bead-one-compound combinatorial library synthesis, screening and identification of substrate motifs of Protein Kinases
Student: Ekama Onofiok
Mentor: Kit S. Lam
Department of Internal Medicine
The one-bead-one-compound (OBOC) combinatorial library method is characterized by randomized on-resin-bead library synthesis and screening, where each bead contains a different compound entity. This method is a highly efficient approach to identifying ligands for a large number of biological targets and thus, has been used to identify peptide substrates for Etk- a tyrosine kinase expressed in endothelial, hematopoetic, epithelial cells, and overexpressed in prostrate cancers. Also, using this method, an encoded peptidomimetic library was designed and synthesized, based on known substrate motifs for certain protein tyrosine kinases (PTKs) in an effort to develop potent pseudo-substrate inhibitors for these PTKs.