X Yu

BCL-6 Represses Genes that Function in Lymphocyte Differentiation, Inflammation, and Cell Cycle Control

BCL-6, a transcriptional repressor frequently translocated in lymphomas, regulates germinal center B cell differentiation and inflammation. DNA microarray screening identified genes repressed by BCL-6, including many lymphocyte activation genes, suggesting that BCL-6 modulates B cell receptor signals. BCL-6 repression of two chemokine genes, MIP-1alpha and IP-10, may also attenuate inflammatory responses. Blimp-1, another BCL-6 target, is important for plasmacytic differentiation. Since BCL-6 expression is silenced in plasma cells, repression of blimp-1 by BCL-6 may control plasmacytic differentiation. Indeed, inhibition of BCL-6 function initiated changes indicative of plasmacytic differentiation, including decreased expression of c-Myc and increased expression of the cell cycle inhibitor p27kip1. These data suggest that malignant transformation by BCL-6 involves inhibition of differentiation and enhanced proliferation.

Direct Repression of prdm1 by Bcl-6 Inhibits Plasmacytic Differentiation

We have identified two intronic regions of mouse prdm1, the gene encoding B lymphocyte-induced maturation protein-1 (Blimp-1), which confer transcriptional repression in response to Bcl-6. The Bcl-6 response element in intron 5, which is conserved between mice and humans, was studied in detail. It binds Bcl-6 in vitro and was shown by chromatin immunoprecipitation to be occupied by Bcl-6 in vivo. Neither Bcl-6 response element functions as a STAT3-response element, showing that STAT3 does not compete with Bcl-6 at these sites. Bcl-6−/− mice confirm the biological importance of Bcl-6-dependent repression of prdm1. These mice have elevated Ab response, increased Ig-secreting cells, and increased Blimp-1+ cells in spleen following immunization and their splenic B cells show accelerated plasmacytic development in vitro.

In vivo auditory brain mapping in mice with Mn-enhanced MRI

There are currently no noninvasive imaging methods available for auditory brain mapping in mice, despite the increasing use of genetically engineered mice to study auditory brain development and hearing loss. We developed a manganese-enhanced MRI (MEMRI) method to map regions of accumulated sound-evoked activity in awake, normally behaving mice. To demonstrate its utility for high-resolution (100-μm) brain mapping, we used MEMRI to show the tonotopic organization of the mouse inferior colliculus. To test its efficacy in an experimental setting, we acquired data from mice experiencing unilateral conductive hearing loss at different ages. Larger and persistent changes in auditory brainstem activity resulted when hearing loss occurred before the onset of hearing, showing that early hearing loss biases the response toward the functional ear. Thus, MEMRI provides a sensitive and effective method for mapping the mouse auditory brainstem and has great potential for a range of functional neuroimaging studies in normal and mutant mice.

Cellular MRI contrast via coexpression of transferrin receptor and ferritin.

Recently there has been growing interest in the development and use of iron‐based contrast agents for cellular imaging with MRI. In this study we investigated coexpression of the transferrin receptor and ferritin genes to induce cellular contrast in a biological system. Expression of transgenic human transferrin receptor and human ferritin H‐subunit was induced in a stably transfected mouse neural stem cell line. When grown in iron‐rich medium, the transgenic cells accumulated significantly more iron than control cells, with a trend toward an increase in reactive oxygen species, but no detrimental effects on cell viability. This cellular iron significantly increased the transverse relaxivities, R2 and R  2* , at 1.5 T and 7 T. By comparing measurements in the same cell samples at 1.5 T and 7 T, we confirmed the expected increase in relaxivity with increasing field strength. Finally, supplemented transgenic cells transplanted into mouse brain demonstrated increased contrast with surrounding neural tissue on T  2* ‐weighted MR brain images compared to controls. These results indicate that dual expression of proteins at different critical points in the iron metabolism pathway may improve cellular contrast without compromising cell viability. Magn Reson Med, 2006.

Large-scale reorganization of the tonotopic map in mouse auditory midbrain revealed by MRI

The cortex is thought to be the primary site of sensory plasticity, particularly during development. Here, we report that large-scale reorganization of the mouse auditory midbrain tonotopic map is induced by a specific sound-rearing environment consisting of paired low- (16 kHz) and high-frequency (40 kHz) tones. To determine the potential for plasticity in the mouse auditory midbrain, we used manganese-enhanced MRI to analyze the midbrain tonotopic maps of control mice during normal development and mice reared in the two-tone (16 + 40 kHz) environment. We found that the tonotopic map emerged during the third postnatal week in normal mice. Before 3 weeks, a larger percentage of auditory midbrain responded to each of the suprathreshold test frequencies, despite the fact that the primary afferent projections are in place even before hearing onset. By 3 weeks, the midbrain tonotopic map of control mice was established, and manganese-enhanced MRI showed a clear separation between the 16- and 40-kHz responses. Two-tone rearing dramatically altered the appearance of these discrete frequency-specific responses. A significant volume of the auditory midbrain became responsive to both rearing frequencies, resulting in a large-scale reorganization of the tonotopic map. These results indicate that developmental plasticity occurs on a much greater scale than previously appreciated in the mammalian auditory midbrain.

Compensatory IKKα activation of classical NF-κB signaling during IKKβ inhibition identified by an RNA interference sensitization screen

A subtype of diffuse large B-cell lymphoma (DLBCL), termed activated B-cell-like (ABC) DLBCL, depends on constitutive nuclear factor-κB (NF-κB) signaling for survival. Small molecule inhibitors of IκB kinase β (IKKβ), a key regulator of the NF-κB pathway, kill ABC DLBCL cells and hold promise for the treatment of this lymphoma type. We conducted an RNA interference genetic screen to investigate potential mechanisms of resistance of ABC DLBCL cells to IKKβ inhibitors. We screened a library of small hairpin RNAs (shRNAs) targeting 500 protein kinases for shRNAs that would increase the killing of an ABC DLBCL cell line in the presence of a small molecule IKKβ inhibitor. Two independent shRNAs targeting IKKα synergized with the IKKβ inhibitor to kill three different ABC DLBCL cell lines but were not toxic by themselves. Surprisingly, IKKα shRNAs blocked the classical rather than the alternative NF-κB pathway in ABC DLBCL cells, as judged by inhibition of IκBα phosphorylation. IKKα shRNA toxicity was reversed by coexpression of wild-type but not kinase inactive forms of IKKα, suggesting that IKKα may directly phosphorylate IκBα under conditions of IKKβ inhibition. In models of physiologic NF-κB pathway activation by CARD11 or tumor necrosis factor-α, compensatory IKKα activity was also observed with IKKβ inhibition. These results suggest that therapy for ABC DLBCL may be improved by targeting both IKKα and IKKβ, possibly through CARD11 inhibition.

Statistical mapping of sound-evoked activity in the mouse auditory midbrain using Mn-enhanced MRI

Manganese-enhanced MRI (MEMRI) has been developed to image brain activity in small animals, including normal and genetically modified mice. Here, we report the use of a MEMRI-based statistical parametric mapping method to analyze sound-evoked activity in the mouse auditory midbrain, the inferior colliculus (IC). Acoustic stimuli with defined frequency and amplitude components were shown to activate and enhance neuronal ensembles in the IC. These IC activity patterns were analyzed quantitatively using voxel-based statistical comparisons between groups of mice with or without sound stimulation. Repetitive 40-kHz pure tone stimulation significantly enhanced ventral IC regions, which was confirmed in the statistical maps showing active regions whose volumes increased in direct proportion to the amplitude of the sound stimuli (65 dB, 77 dB, and 89 dB peak sound pressure level). The peak values of the activity-dependent MEMRI signal enhancement also increased from 7% to 20% for the sound amplitudes employed. These results demonstrate that MEMRI statistical mapping can be used to analyze both the 3D spatial patterns and the magnitude of activity evoked by sound stimuli carrying different energy. This represents a significant advance in the development of MEMRI for quantitative and unbiased analysis of brain function in the deep brain nuclei of mice.

Direct imaging of macrovascular and microvascular contributions to BOLD fMRI in layers IV-V of the rat whisker-barrel cortex

The spatiotemporal characteristics of the hemodynamic response to increased neural activity were investigated at the level of individual intracortical vessels using BOLD-fMRI in a well-established rodent model of somatosensory stimulation at 11.7 T. Functional maps of the rat barrel cortex were obtained at 150 × 150 × 500 μm spatial resolution every 200 ms. The high spatial resolution allowed separation of active voxels into those containing intracortical macro vessels, mainly vein/venules (referred to as macrovasculature), and those enriched with arteries/capillaries and small venules (referred to as microvasculature) since the macro vessel can be readily mapped due to the fast T2 decay of blood at 11.7 T. The earliest BOLD response was observed within layers IV-V by 0.8s following stimulation and encompassed mainly the voxels containing the microvasculature and some confined macrovasculature voxels. By 1.2s, the BOLD signal propagated to the macrovasculature voxels where the peak BOLD signal was 2-3 times higher than that of the microvasculature voxels. The BOLD response propagated in individual venules/veins far from neuronal sources at later times. This was also observed in layers IV-V of the barrel cortex after specific stimulation of separated whisker rows. These results directly visualized that the earliest hemodynamic changes to increased neural activity occur mainly in the microvasculature and spread toward the macrovasculature. However, at peak response, the BOLD signal is dominated by penetrating venules even at layers IV-V of the cortex.

Deciphering laminar-specific neural inputs with line-scanning fMRI

Using a line-scanning method during functional magnetic resonance imaging (fMRI), we obtained high temporal (50-ms) and spatial (50-μm) resolution information along the cortical thickness and showed that the laminar position of fMRI onset coincides with distinct neural inputs in rat somatosensory and motor cortices. This laminar-specific fMRI onset allowed us to identify the neural inputs underlying ipsilateral fMRI activation in the barrel cortex due to peripheral denervation-induced plasticity.

Thalamocortical Inputs Show Post-Critical Period Plasticity

Experience-dependent plasticity in the adult brain has clinical potential for functional rehabilitation following central and peripheral nerve injuries. Here, plasticity induced by unilateral infraorbital (IO) nerve resection in 4-week-old rats was mapped using MRI and synaptic mechanisms were elucidated by slice electrophysiology. Functional MRI demonstrates a cortical potentiation compared to thalamus 2 weeks after IO nerve resection. Tracing thalamocortical (TC) projections with manganese-enhanced MRI revealed circuit changes in the spared layer 4 (L4) barrel cortex. Brain slice electrophysiology revealed TC input strengthening onto L4 stellate cells due to an increase in postsynaptic strength and the number of functional synapses. This work shows that the TC input is a site for robust plasticity after the end of the previously defined critical period for this input. Thus, TC inputs may represent a major site for adult plasticity in contrast to the consensus that adult plasticity mainly occurs at cortico-cortical connections.