Faculty Bibliography

Endothelial cells line blood vessels and coordinate many aspects of vascular biology. More recent work has shown that endothelial cells provide a key niche in vivo for neural stem cells. In vitro, endothelial cells secrete a factor that expands neural stem cells while inhibiting their differentiation. Here, we show that a transformed mouse endothelial cell line (bEnd.3) maintains human pluripotent stem cells in an undifferentiated state. bEnd.3 cells have a practical advantage over mouse embryonic fibroblasts for pluripotent stem cell maintenance since they can be expanded in vitro and engineered to express genes of interest. We demonstrate this capability by producing fluorescent and drug-resistant feeder cells. Further, we show that bEnd.3 secretes an activity that maintains human embryonic stem cells without direct contact.

Axonal degeneration after traumatic brain injury and nerve compression is considered a common underlying cause of temporary as well as permanent disability. Because a proper functioning of neural network requires phase coherence of all components, even subtle changes in circuitry may lead to network failure. However, it is still not possible to determine which axons will recover or degenerate after injury. Several groups have studied the pressure threshold for axonal injury within a nerve, but difficulty accessing the injured region; insufficient imaging methods and the extremely small dimensions involved have prevented the evaluation of the response of individual axons to injury. We combined microfluidics with atomic force microscopy and in vivo imaging to estimate the threshold force required to 1), uncouple axonal transport without impairing axonal survival, and 2), compromise axonal survival in both individual and bundled axons. We found that rat hippocampal axons completely recover axonal transport with no detectable axonal loss when compressed with pressures up to 65 +/- 30 Pa for 10 min, while dorsal root ganglia axons can resist to pressures up to 540 +/- 220 Pa. We investigated the reasons for the differential susceptibility of hippocampal and DRG axons to mechanical injury and estimated the elasticity of live axons. We found that dorsal root ganglia axons have a 20% lower elastic modulus than hippocampal axons. Our results emphasize the importance of the integrity of the axonal cytoskeleton in deciding the axonal fate after damage and open up new avenues to improve injury diagnosis and to identify ways to protect axons.

Gene expression programs that regulate the abundance of the chaperone BiP adapt the endoplasmic reticulum (ER) to unfolded protein load. However, such programs are slow compared with physiological fluctuations in secreted protein synthesis. While searching for mechanisms that fill this temporal gap in coping with ER stress, we found elevated levels of adenosine diphosphate (ADP)-ribosylated BiP in the inactive pancreas of fasted mice and a rapid decline in this modification in the active fed state. ADP ribosylation mapped to Arg470 and Arg492 in the substrate-binding domain of hamster BiP. Mutations that mimic the negative charge of ADP-ribose destabilized substrate binding and interfered with interdomain allosteric coupling, marking ADP ribosylation as a rapid posttranslational mechanism for reversible inactivation of BiP. A kinetic model showed that buffering fluctuations in unfolded protein load with a recruitable pool of inactive chaperone is an efficient strategy to minimize both aggregation and costly degradation of unfolded proteins.

Despite their usefulness as fluorophores and synthetic precursors, efficient and reliable routes to coumarin-8-carbaldehydes are lacking. We describe here a high-yielding continuous flow synthesis that requires no manual intermediate purification or work-up, giving access to multigram quantities of the aldehyde product.

The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, phospholamban, are essential components of cardiac contractility. Phospholamban modulates contractility by inhibiting SERCA, and this process is dynamically regulated by beta-adrenergic stimulation and phosphorylation of phospholamban. Herein we reveal mechanistic insight into how four hereditary mutants of phospholamban, Arg(9) to Cys, Arg(9) to Leu, Arg(9) to His, and Arg(14) deletion, alter regulation of SERCA. Deletion of Arg(14) disrupts the protein kinase A recognition motif, which abrogates phospholamban phosphorylation and results in constitutive SERCA inhibition. Mutation of Arg(9) causes more complex changes in function, where hydrophobic substitutions such as cysteine and leucine eliminate both SERCA inhibition and phospholamban phosphorylation, whereas an aromatic substitution such as histidine selectively disrupts phosphorylation. We demonstrate that the role of Arg(9) in phospholamban function is multifaceted: it is important for inhibition of SERCA, it increases the efficiency of phosphorylation, and it is critical for protein kinase A recognition in the context of the phospholamban pentamer. Given the synergistic consequences on contractility, it is not surprising that the mutants cause lethal, hereditary dilated cardiomyopathy.

Whole-mount RNA in situ hybridizations with digoxigenin-conjugated probes and alkaline phosphatase biochemistry have been used widely for many years to map expression pattern domains in the Drosophila embryo. To capitalize on the number of molecular markers in the central nervous system (CNS) and to enable expression analysis at the single-cell level, fluorescence in situ hybridization procedures are becoming standard. This protocol describes methods for the simultaneous detection of RNA and protein using fluorescence in Drosophila embryos. It uses the tyramide signal amplication (TSA) system from PerkinElmer to amplify a horseradish peroxidase (HRP) signal. By combining this technology with an HRP-conjugated antidigoxigenin antibody, we can detect standard antidigoxigenin RNA probes fluorescently.

BACKGROUND: Angelman syndrome (AS) is a human neuropsychiatric disorder associated with autism, mental retardation, motor abnormalities, and epilepsy. In most cases, AS is caused by the deletion of the maternal copy of UBE3A gene, which encodes the enzyme ubiquitin ligase E3A, also termed E6-AP. A mouse model of AS has been generated and these mice exhibit many of the observed neurological alterations in humans. Because of clinical and neuroanatomical similarities between AS and schizophrenia, we examined AS model mice for alterations in the neuregulin-ErbB4 pathway, which has been implicated in the pathophysiology of schizophrenia. We focused our studies on the hippocampus, one of the major brain loci impaired in AS mice. METHODS: We determined the expression of neuregulin 1 and ErbB4 receptors in AS mice and wild-type littermates (ages 10-16 weeks) and studied the effects of ErbB inhibition on long-term potentiation in hippocampal area cornu ammonis 1 and on hippocampus-dependent contextual fear memory. RESULTS: We observed enhanced neuregulin-ErbB4 signaling in the hippocampus of AS model mice and found that ErbB inhibitors could reverse deficits in long-term potentiation, a cellular substrate for learning and memory. In addition, we found that an ErbB inhibitor enhanced long-term contextual fear memory in AS model mice. CONCLUSIONS: Our findings suggest that neuregulin-ErbB4 signaling is involved in synaptic plasticity and memory impairments in AS model mice, suggesting that ErbB inhibitors have therapeutic potential for the treatment of AS.

Genes dedicated to killing cells must have evolved because of their positive effects on organismal survival. Positive functions of apoptotic genes have been well established in a large number of biological contexts, including their role in eliminating damaged and potentially cancerous cells. More recently, evidence has suggested that proapoptotic proteins-mostly caspases-can induce proliferation of neighboring surviving cells to replace dying cells. This process, that we will refer to as "apoptosis-induced proliferation," may be critical for stem cell activity and tissue regeneration. Depending on the caspases involved, at least two distinct types of apoptosis-induced proliferation can be distinguished. One of these types have been studied using a model in which cells have initiated cell death, but are prevented from executing it because of effector caspase inhibition, thereby generating "undead" cells that emit persistent mitogen signaling and overgrowth. Such conditions are likely to contribute to certain forms of cancer. In this review, we summarize the current knowledge of apoptosis-induced proliferation and discuss its relevance for tissue regeneration and cancer.

As neurons age, their survival depends on eliminating a growing burden of damaged, potentially toxic proteins and organelles-a capability that declines owing to aging and disease factors. Here, we review the two proteolytic systems principally responsible for protein quality control in neurons and their important contributions to Alzheimer disease pathogenesis. In the first section, the discovery of paired helical filament ubiquitination is described as a backdrop for discussing the importance of the ubiquitin-proteasome system in Alzheimer disease. In the second section, we review the prominent involvement of the lysosomal system beginning with pathological endosomal-lysosomal activation and signaling at the very earliest stages of Alzheimer disease followed by the progressive failure of autophagy. These abnormalities, which result in part from Alzheimer-related genes acting directly on these lysosomal pathways, contribute to the development of each of the Alzheimer neuropathological hallmarks and represent a promising therapeutic target.

The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase TrkB serve important regulatory roles for multiple aspects of the biology of neurons including cell death, survival, growth, differentiation, and plasticity. Regulation of the local availability of BDNF/TrkB at distinct subcellular domains such as soma, dendrites, axons, growth cones, nerve terminals, and spines appears to contribute to their specific functions. In view of the variance in size and shape of neurons and their compartments, previous quantitative studies of the BDNF/TrkB protein and mRNA lacked a robust normalization procedure. To overcome this problem, we have established methods that use immunofluorescence detection of alpha-tubulin as a normalization factor for the quantitative analysis of protein and mRNA in primary rat cortical and striatal neurons in culture. The efficacy of this approach is demonstrated by studying the dynamic distribution of proteins and mRNA at different growth stages or conditions. Treatment of cultured neurons with KCl resulted in increased levels of TrkB protein, reduced levels of BDNF mRNA (composite of multiple transcripts) and a slight reduction in BDNF protein levels in the dendrites from the cortex. The KCl treatment also lowered the percentage of BDNF and TrkB proteins in the soma indicative of protein transport. Finally, analysis of the rat cortical and striatal neurons demonstrated comparable or even higher levels of BDNF/TrkB protein and BDNF mRNA in the neurons from the striatum. Thus, in contrast to previous observations made in vivo, striatal neurons are capable of synthesizing BDNF mRNA when cultured in growth media in vitro. The analytical approach presented here provides a detailed understanding of BDNF/TrkB levels in response to a variety of neuronal activities. Our methods could be used broadly, including applications in cell and tissue cytometry, to yield accurate quantitative data of gene expression in cellular and subcellular contexts. (c) 2012 International Society for Advancement of Cytometry.