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In JoVE (1)
Other Publications (13)
- Journal of the American Chemical Society
- The Journal of Biological Chemistry
- Bioorganic & Medicinal Chemistry Letters
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Molecular Neurodegeneration
- Neuroscience Letters
- Molecular and Cellular Pharmacology
- ACS Chemical Biology
- Journal of the American Chemical Society
- Chemical Communications (Cambridge, England)
- Journal of the American Chemical Society
- Analytical Chemistry
- Chemical Communications (Cambridge, England)
Articles by Jennifer L. Furman in JoVE
JoVE Neuroscience
Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology
1Graduate Center for Gerontology, University of Kentucky College of Public Health, 2Department of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, 3Sanders-Brown Center on Aging, University of Kentucky College of Medicine
This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer’s disease.
Other articles by Jennifer L. Furman on PubMed
Site-specific Detection of DNA Methylation Utilizing MCpG-SEER
Currently there are no direct methods for the sequence-specific detection of DNA-methylation at CpG dinucleotides, which provide a possible diagnostic marker for cancer. Toward this goal, we present a methodology termed mCpG-SEquence Enabled Reassembly (mCpG-SEER) of proteins utilizing a split green fluorescent protein (GFP) tethered to specific DNA recognition elements. Our system, mCpG-SEER, employs a zinc-finger attached to one-half of GFP to target a specific sequence of dsDNA, while a methyl-CpG binding domain protein attached to the complementary half of GFP targets an adjacent methylated CpG dinucleotide site. We demonstrate that the presence of both DNA sites is necessary for the reassembly and concomitant fluorescence of the reassembled GFP. We further show that the GFP-dependent fluorescence reaches a maximum when the methyl-CpG and zinc-finger sites are separated by two base pairs and the fluorescence signal is linear to 5 pmol of methylated target DNA. Finally, the specificity of this reporter system, mCpG-SEER, was found to be >40-fold between a methylated versus a nonmethylated CpG target site.
Interleukin-1beta-dependent Signaling Between Astrocytes and Neurons Depends Critically on Astrocytic Calcineurin/NFAT Activity
Interleukin-1beta (IL-1beta) and the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, have each been shown to play an important role in neuroinflammation. However, whether these signaling molecules interact to coordinate immune/inflammatory processes and neurodegeneration has not been investigated. Here, we show that exogenous application of IL-1beta (10 ng/ml) recruited calcineurin/NFAT (nuclear factor of activated T cells) activation in primary astrocyte-enriched cultures within minutes, through a pathway involving IL-1 receptors and L-type Ca(2+) channels. Adenovirus-mediated delivery of the NFAT inhibitor, VIVIT, suppressed the IL-1beta-dependent induction of several inflammatory mediators and/or markers of astrocyte activation, including tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor, and vimentin. Expression of an activated form of calcineurin in one set of astrocyte cultures also triggered the release of factors that, in turn, stimulated NFAT activity in a second set of "naive" astrocytes. This effect was prevented when calcineurin-expressing cultures co-expressed VIVIT, suggesting that the calcineurin/NFAT pathway coordinates positive feedback signaling between astrocytes. In the presence of astrocytes and neurons, 48-h delivery of IL-1beta was associated with several excitotoxic effects, including NMDA receptor-dependent neuronal death, elevated extracellular glutamate, and hyperexcitable synaptic activity. Each of these effects were reversed or ameliorated by targeted delivery of VIVIT to astrocytes. IL-1beta also caused an NFAT-dependent reduction in excitatory amino acid transporter levels, indicating a possible mechanism for IL-1beta-mediated excitotoxicity. Taken together, the results have potentially important implications for the propagation and maintenance of neuroinflammatory signaling processes associated with many neurodegenerative conditions and diseases.
Systematic Evaluation of Split-fluorescent Proteins for the Direct Detection of Native and Methylated DNA
In order to directly detect nucleic acid polymers, we have designed biosensors comprising sequence-specific DNA binding proteins tethered to split-reporter proteins, which generate signal upon binding a predetermined nucleic acid target, in an approach termed SEquence-Enabled Reassembly (SEER). Herein we demonstrate that spectroscopically distinct split-fluorescent protein variants, GFPuv, EGFP, Venus, and mCherry, function effectively in the SEER system, providing sensitive DNA detection and the ability to simultaneously detect two target oligonucleotides. Additionally, a methylation-specific SEER-Venus system was generated, which was found to clearly distinguish between methylated versus non-methylated target DNA. These results will aid in refinement of the SEER system for the detection of user defined nucleic acid sequences and their chemical modifications as they relate to human disease.
Cognitive Decline in Alzheimer's Disease is Associated with Selective Changes in Calcineurin/NFAT Signaling
Upon activation by calcineurin, the nuclear factor of activated T-cells (NFAT) translocates to the nucleus and guides the transcription of numerous molecules involved in inflammation and Ca(2+) dysregulation, both of which are prominent features of Alzheimer's disease (AD). However, NFAT signaling in AD remains relatively uninvestigated. Using isolated cytosolic and nuclear fractions prepared from rapid-autopsy postmortem human brain tissue, we show that NFATs 1 and 3 shifted to nuclear compartments in the hippocampus at different stages of neuropathology and cognitive decline, whereas NFAT2 remained unchanged. NFAT1 exhibited greater association with isolated nuclear fractions in subjects with mild cognitive impairment (MCI), whereas NFAT3 showed a strong nuclear bias in subjects with severe dementia and AD. Similar to NFAT1, calcineurin-Aalpha also exhibited a nuclear bias in the early stages of cognitive decline. But, unlike NFAT1 and similar to NFAT3, the nuclear bias for calcineurin became more pronounced as cognition worsened. Changes in calcineurin/NFAT3 were directly correlated to soluble amyloid-beta (Abeta((1-42))) levels in postmortem hippocampus, and oligomeric Abeta, in particular, robustly stimulated NFAT activation in primary rat astrocyte cultures. Oligomeric Abeta also caused a significant reduction in excitatory amino acid transporter 2 (EAAT2) protein levels in astrocyte cultures, which was blocked by NFAT inhibition. Moreover, inhibition of astrocytic NFAT activity in mixed cultures ameliorated Abeta-dependent elevations in glutamate and neuronal death. The results suggest that NFAT signaling is selectively altered in AD and may play an important role in driving Abeta-mediated neurodegeneration.
Expression of SORL1 and a Novel SORL1 Splice Variant in Normal and Alzheimers Disease Brain
ABSTRACT:
Disparate Effects of Serum on Basal and Evoked NFAT Activity in Primary Astrocyte Cultures
In astrocytes, the Ca(2+)-dependent protein phosphatase calcineurin (CN) strongly regulates neuro-immune/inflammatory cascades through activation of the transcription factor, nuclear factor of activated T cells (NFAT). While primary cell cultures provide a useful model system for investigating astrocytic CN/NFAT signaling, variable results may arise both within and across labs because of differences in culture conditions. Here, we determined the extent to which serum and cell confluency affect basal and evoked astrocytic NFAT activity in primary cortical astrocyte cultures. Cells were grown to either approximately 50% or >90% confluency, pre-loaded with an NFAT-luciferase reporter construct, and maintained for 16 h in medium with or without 10% fetal bovine serum (FBS). NFAT-dependent luciferase expression was then measured 5h after treatment with vehicle alone to assess basal NFAT activity, or with Ca(2+) mobilizers and IL-1 beta to assess evoked activity. The results revealed significantly higher levels of basal NFAT activity in FBS-containing medium, regardless of cell confluency. Conversely, evoked NFAT activation was significantly lower in serum-containing medium, with an even greater inhibition observed in confluent cultures. Application of 10% FBS to serum-free astrocyte cultures quickly evoked a roughly seven-fold increase in NFAT activity that was significantly reduced by co-delivery of neutralizing agents for IL-1 beta, TNFalpha, and/or IFN gamma, suggesting that serum occludes evoked NFAT activation through a cytokine-based mechanism. Together, the results demonstrate that the presence of serum and cell confluency have a major impact on CN/NFAT signaling in primary astrocyte cultures and therefore must be taken into consideration when using this model system.
NFATs and Alzheimer's Disease
Nuclear factor of activated T cells (NFAT) is a transcription factor that translocates from cytosol to nucleus following dephosphorylation by the Ca(2+)/calmodulin dependent protein phosphatase calcineurin (CN). In nervous tissue, aberrant CN signaling is increasingly linked to a variety of pathologic features associated with Alzheimer's disease (AD), including synaptic dysfunction, glial activation, and neuronal death. Consistent with this linkage, our recent work on postmortem human hippocampal tissue discovered increased nuclear accumulation of select NFAT isoforms at different stages of AD. Some of these changes occurred at the early stages of the disease process and/or paralleled diminishing cognitive status. In addition, inhibition of astrocytic NFAT activity in primary cultures of neurons and glia dampened glutamate levels and alleviated neuronal death in response to pathogenic amyloid-β peptides. In this article, we discuss our recent findings and expand upon the possible isoform specific contributions of NFATs to the progression of AD. We also consider the possible benefits of using NFAT inhibitors to treat AD and other neurodegenerative disorders, as well.
A General Approach for Receptor and Antibody-targeted Detection of Native Proteins Utilizing Split-luciferase Reassembly
The direct detection of native proteins in heterogeneous solutions remains a challenging problem. Standard methodologies rely on a separation step to circumvent nonspecific signal generation. We hypothesized that a simple and general method for the detection of native proteins in solution could be achieved through ternary complexation, where the conditional signal generation afforded by split-protein reporters could be married to the specificity afforded by either native receptors or specific antibodies. Toward this goal, we describe a solution phase split-luciferase assay for native protein detection, where we fused fragmented halves of firefly luciferase to separate receptor fragments or single-chain antibodies, allowing for conditional luciferase complementation in the presence of several biologically significant protein targets. To demonstrate the utility of this strategy, we have developed and validated assay platforms for the vascular endothelial growth factor, the gp120 coat protein from HIV-1, and the human epidermal growth factor receptor 2 (HER2), a marker for breast cancer. The specificities of the recognition elements, CD4 and the 17b single-chain antibody, employed in the gp120 sensor allowed us to parse gp120s from different clades. Our rationally designed HER2 sensing platform was capable of discriminating between HER2 expression levels in several tumor cell lines. In addition, luminescence from reassembled luciferase was linear across a panel of cell lines with increasing HER2 expression. We envision that the proof of principle studies presented herein may allow for the potential detection of a broad range of biological analytes utilizing ternary split-protein systems.
Toward a General Approach for RNA-templated Hierarchical Assembly of Split-proteins
The ability to conditionally turn on a signal or induce a function in the presence of a user-defined RNA target has potential applications in medicine and synthetic biology. Although sequence-specific pumilio repeat proteins can target a limited set of ssRNA sequences, there are no general methods for targeting ssRNA with designed proteins. As a first step toward RNA recognition, we utilized the RNA binding domain of argonaute, implicated in RNA interference, for specifically targeting generic 2-nucleotide, 3' overhangs of any dsRNA. We tested the reassembly of a split-luciferase enzyme guided by argonaute-mediated recognition of newly generated nucleotide overhangs when ssRNA is targeted by a designed complementary guide sequence. This approach was successful when argonaute was utilized in conjunction with a pumilio repeat and expanded the scope of potential ssRNA targets. However, targeting any desired ssRNA remained elusive as two argonaute domains provided minimal reassembled split-luciferase. We next designed and tested a second hierarchical assembly, wherein ssDNA guides are appended to DNA hairpins that serve as a scaffold for high affinity zinc fingers attached to split-luciferase. In the presence of a ssRNA target containing adjacent sequences complementary to the guides, the hairpins are brought into proximity, allowing for zinc finger binding and concomitant reassembly of the fragmented luciferase. The scope of this new approach was validated by specifically targeting RNA encoding VEGF, hDM2, and HER2. These approaches provide potentially general design paradigms for the conditional reassembly of fragmented proteins in the presence of any desired ssRNA target.
Profiling Small Molecule Inhibitors Against Helix-receptor Interactions: the Bcl-2 Family Inhibitor BH3I-1 Potently Inhibits P53/hDM2
We validate a practical methodology for the rapid profiling of small molecule inhibitors of protein-protein interactions. We find that a well known BH3 family inhibitor can potently inhibit the p53/hDM2 interaction.
Turn-on DNA Damage Sensors for the Direct Detection of 8-oxoguanine and Photoproducts in Native DNA
The integrity of the genetic information in all living organisms is constantly threatened by a variety of endogenous and environmental insults. To counter this risk, the DNA-damage response is employed for repairing lesions and maintaining genomic integrity. However, an aberrant DNA-damage response can potentially lead to genetic instability and mutagenesis, carcinogenesis, or cell death. To directly monitor DNA damage events in the context of native DNA, we have designed two new sensors utilizing genetically fragmented firefly luciferase (split luciferase). The sensors are comprised of a methyl-CpG binding domain (MBD) attached to one fragment of split luciferase for localizing the sensor to DNA (50-80% of the CpG dinucleotide sites in the genome are symmetrically methylated at cytosines), while a damage-recognition domain is attached to the complementary fragment of luciferase to probe adjacent nucleotides for lesions. Specifically, we utilized oxoguanine glycosylase 1 (OGG1) to detect 8-oxoguanine caused by exposure to reactive oxygen species and employed the damaged-DNA binding protein 2 (DDB2) for detection of pyrimidine dimer photoproducts induced by UVC light. These two sensors were optimized and validated using oligonucleotides, plasmids, and mammalian genomic DNA, as well as HeLa cells that were systematically exposed to a variety of environmental insults, demonstrating that this methodology utilizing MBD-directed DNA localization provides a simple, sensitive, and potentially general approach for the rapid profiling of specific chemical modifications associated with DNA damage and repair.
Evaluating the Global CpG Methylation Status of Native DNA Utilizing a Bipartite Split-luciferase Sensor
Epigenetic modifications play an essential role in the regulation of gene expression and ultimately cell fate. Methylation of cytosine at CpG dinucleotides (mCpG) is an important epigenetic mark that has been correlated with cancer when present at promoter sites of tumor suppressor genes. To develop a rapid methodology for the direct assessment of global levels of DNA methylation, we first interrogated the methyl-CpG binding domains (MBDs), the Kaiso family of Cys(2)-His(2) zinc fingers, and an SET- and RING-associated domain using a split-luciferase reassembly methodology. We identified MBD1 as the most selective domain for the discrimination between mCpG and CpG sites with over 90-fold selectivity. Utilizing a bipartite strategy, we constructed a purely methylation-dependent bipartite sensor for the direct detection of global levels of DNA methylation by attaching MBD1 domains to each of the split-luciferase halves. This new sensor was validated for the direct determination of genomic DNA methylation levels in in vitro studies without any intervening chemical or enzymatic processing of DNA. Finally, we demonstrated that this bipartite sensor can be utilized for monitoring dose-dependent changes in global levels of methylation in DNA from HeLa cells challenged with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor.
A Turn-on Split-luciferase Sensor for the Direct Detection of Poly(ADP-ribose) As a Marker for DNA Repair and Cell Death
Designed sensors comprising split-firefly luciferase conjugated to tandem poly(ADP-ribose) binding domains allow for the direct solution phase detection of picogram quantities of PAR and for monitoring temporal changes in poly(ADP-ribosyl)ation events in mammalian cells.
