Research

Project 1. Development of new tools for probing mitochondrial activity in cells and living animals

Introduction

Mitochondria are essential organelles that provide eukaryotic cells with energy in the form of ATP generated through aerobic respiration. Electrons harvested from the oxidation of carbon food sources are utililized to pump protons across the inner membrane and store the energy in a proton gradient, which is then used to produce ATP through chemiosmosis. Aside from this critical role of ATP production, mitochondria are also important in multiple biological processes including cell differentiation,[1-2] cell cycle control,[1-4] cell survival,1-4 neuronal protection,[5-7] and aging.[8-9] Mitochondrial dysfunction contributes to a remarkably wide range of human diseases including cancer,[10] Alzheimer’s disease,[5-7] Parkinson’s disease,[11] diabetes,[12-13] ischemia perfusion injury,[14] steatohepatitis,[15] sepsis,[16] Huntington’s disease,[17] and many others.[18-19] As more information associating mitochondrial dysfunction with human diseases emerges, the development of new tools to interrogate this important organelle becomes increasingly important.

Figure 1

Figure 1. Image of labeled mitochondria. HEK293 cells were transfected with mitoGFP-1 marker, that is specific for mitochondria (courtesy of Dr. Fen Huang).

Maintenance of the mitochondrial membrane potential (ΔΨm) is fundamental for the normal performance, differentiation, and survival of cells. In nearly all cases where mitochondrial malfunction contributes to a disease, mitochondrial membrane potential is perturbed.[5-7,10-20]

Although it is unclear whether changes in the mitochondrial membrane potential are a primary cause or a secondary event, it is universally agreed that changes in ΔΨm directly reflect the cell life to death transition.3-4

Therefore, measurements of mitochondrial membrane potential should provide important information about the molecular mechanisms controlling cell functions and are of considerable interest for biomedical research and drug development.

Aim 1.1. Developing new techniques for quantification of mitochondrial membrane potential (ΔΨm) in living cells and animals
A major problem in studying mitochondrial potential in living cells and animals is the lack of specific tools. The current methods for measurement of ΔΨm are based on fluorescence-activated cell sorting (FACS) and confocal microscopy, and rely on cationic lipophilic fluorescent dyes (Fig. 2). These dyes, which are membrane permeable, are generally thought to accumulate specifically in active mitochondria in proportion to the magnitude of ΔΨm.[21-30] Potential-dependent accumulation of these dyes by mitochondria results in significant emission wavelength shift due to formation of dye aggregates. For example, in case of the dye JC-1, the fluorescence shifts from 527 nm to 590 nm upon aggregation. This ratio of “green” to “red” fluorescence excited at 488 nm is used to detect ΔΨm changes.[21-22] These dyes have been actively used to assess changes in ΔΨm parameter in a variety of different cell lines.[23-25] Nevertheless, their use as sensors of ΔΨm is limited because they have been shown to undergo self-quenching upon accumulation in the mitochondrial matrix.[21,26-29]

Another limitation is nonspecific binding of the dyes to cytosolic macromolecules and to both inner and outer mitochondrial membranes, which contribute to the overall background signal.[26-30] Moreover, the readout from these dyes is dependent on intracellular pH and the protein/membrane content, rendering comparisons between different cell lines impossible.[28-30] Another problem limiting application of these current probes in living systems is their high toxicity [22,26-28] and enhanced efflux by p-glycoprotein transporter.[31]

Figure 2 Figure 2. Current state of the art for measuring ΔΨm. JC-1 dye has been a “golden standard” for the measurement of ΔΨm during the last decade. The dye is taken up by cells and specifically accumulated inside the mitochondria due to its cationic and lipophilic nature in proportion to the magnitude of ΔΨm. In healthy mitochondria the fluorescence of JC-1 shifts from 527 nm to 590 nm upon aggregation. This ratio of “green” to “red” fluorescence excited at 488 nm is used to detect ΔΨm changes.

Therefore, new techniques that overcome these limitations and allow for reliable measurement of ΔΨm in living cells and animals are of significant benefit to the study of  mitochondria.

Combining chemical biology approaches with high sensitivity optical imaging tools and improved algorithms for data analysis, my lab will exploit the differences in ΔΨm that are associated with various diseases with an eye towards identifying new avenues for clinical imaging and drug development. The new methods described below have potential to overcome the limitations of the existing probes, as well as allow measurements of ΔΨm in significantly more complex biological settings. Since mice are the most common model of human disease, developing techniques for in vivo imaging of ΔΨm in mice could advance our understanding of the biology of mitochondria associated diseases with possible extensions to new therapies.
My lab will initially focus on the application of this method to study the induction of beta-cell dysfunction in diabetes and examine the mitochondrial mechanisms in amyloid beta peptide-induced cerebrovascular degeneration. Both diabetes and diseases related to cerebrovascular degeneration, such as stroke, are among the leading causes of adult morbidity and mortality worldwide [12-13,32-34] and mitochondrial dysfunction is a major cellular event in both of these disease types. [12-13,32-34] We also plan to apply this technology to study functions of uncoupling proteins (UCPs) [20,35-36] that are involved in proton conductance across the inner mitochondrial membrane, and are directly connected to ΔΨm. Recently, there has been significant interest in mitochondria uncoupling as a target for the treatment of obesity.[35-36]

Aim 1.2. Biological applications of mitochondrial membrane potential measurements

1.2.1. Detecting the induction of beta-cell dysfunction in diabetes
Beta-cell death plays a key role in the pathology of diabetes, resulting in partial or complete loss of insulin production.[33-34,57] Mitochondrial dysfunction, which is thought to trigger apoptosis of beta-cells, is the basis for the development of both type-1 and type-2 diabetes, providing a framework for the development of potential therapeutic targets.[13,58] Multiple animal models of diabetes have been developed.[59-61] However, no methods are currently available that would allow the study of beta-cell mediated mitochondrial dysfunction in the context of living animals. My lab will use the new technology described in Aim 1 to further investigate mechanisms leading to induction of beta-cell death and their relation to mitochondrial dysfunction.

1.2.2. Study of the mitochondrial mechanisms in amyloid beta peptide-induced cerebrovascular  degeneration
One of the major consequences of cerebrovascular degeneration is stroke, which is the second leading cause of death and the leading cause of adult disability worldwide.[32] Moreover, cerebrovascular disease and Alzheimer’s disease commonly occur together and are the leading causes of dementia in the elderly.[32] Amyloid beta peptide is a key mediator in neuronal and cerebrovascular degeneration that activates death signaling processes leading to neuronal and non-neuronal cell death in the central nervous system.[32] The major cellular event in amyloid beta peptide activation is mitochondrial dysfunction, but the molecular mechanism of this process that results in cell death still remains to be fully understood.[32] Defining these death signaling processes will enable the development of more specific strategies to slow age-dependent increase in stroke rate. Since mitochondrial dysfunction is directly connected to the loss in ΔΨm,[32] we will use the imaging technology (Aim 1) in several animal disease models that are currently available, with the hope to further illuminate this complicated process.

1.2.3. Studies of mitochondria uncoupling as a target for the treatment of obesity
My lab will further expand the use of the technology described in Aim 1 to study the activity of uncoupling proteins (UCPs). These proteins belong to a family of mitochondrial transport proteins and are involved in proton conductance across the inner mitochondrial membrane, and are directly connected to ΔΨm. [35-36] Emerging evidence suggests that the tissue distribution of UCPs regulates various important biological functions and is implicated in diverse pathologic conditions such as obesity and diabetes,[62] neurodegenerative diseases,[20,63] atherosclerosis,[64] cancer,[65] and sepsis.[66] In addition, recent investigations have shown an important role for these proteins in cerebral stroke[67] as well as lifespan extension.[68] Moreover, recent findings demonstrated that compounds that can activate mitochondrial uncoupling are good potential targets for the treatment of obesity.[68-69]

Despite the importance of UCPs in many biological processes and human diseases, their physiological functions are still poorly understood due to the lack of tools available for their study.[35] Figure 4 summarizes some of the pathological consequences and therapeutic effects of altered expression and activity of different UCPs and shows how many of these functions are still either completely unknown or controversial.

Figure 4

Figure 4. Pathological consequences and therapeutic effects of altered expression and activity of different uncoupling proteins.

My lab will be actively involved in functional studies of these proteins in connection to ΔΨm with the initial focus being on obesity-related disorders. The methods described in Aim 1 could be used to screen compounds in mice that activate uncoupling of UCP1 in brown adipose tissue and therefore are attractive targets for the treatment of obesity.[35-36,68-69] In principle, the method described in Aim 1.2 could be used to identify the mass and activity of brown adipose tissue in humans.

Project 2. New probes for non-invasive imaging of receptor-ligand interactions in live cells and animals

Introduction

Bioorthogonal “click” reactions are reactions whose components react selectively and rapidly with each other in the presence of all the chemical functionality found in living systems. In the past several years these reactions have proven to be essential tools in chemical biology.[41-44] They have been widely used for residue-specific labeling and genetically encoded modifications of proteins, exploration of glycan functions, and analysis of myristoylation, palmitoylation, and farnesylation processes.[41-44]

Aim 2.1. Expanding the diversity of bioorthogonal “click” reactions
Many ”click” reactions have been shown to work in cell lysates and live cells; however, the phosphine-based Staudinger reaction with azides remains the best performing click reaction in vivo.[41,43]  While the Staudinger ligation has been proven to work well in mice, improvements in the rate of this reaction would allow analysis of a wider range of biological processes. Moreover, it would be useful to have a second bioorthogonal reaction, which could be used in concert with the Staudinger ligation to afford imaging of more complex biological processes in living organisms.
After new bioorthogonal reactions are identified, we will use them for several biological applications, including biomolecular labeling and imaging of cellular processes, with a goal of developing improved therapeutics.

Aim 2.2. Application of new bioorthogonal reactions in biological systems
2.2.1. Non-invasive imaging of insulin-receptor binding in live cells and animals

Insulin is a peptide hormone that plays an important role in regulating glucose, fat, and steroid metabolism in the body and is the only effective drug for the treatment of diabetes.[76] The insulin signaling system is central to regulating metabolism, reproduction, growth and development, survival and lifespan, as well as multiple central nervous system functions such as cognition, memory and appetite regulation.[76] Since efforts towards obtaining the crystallographic structure of the insulin-receptor complex are still underway, hundreds of insulin analogs have been made to improve our understanding of the structure-function relationship of this important complex. While the information generated by these biochemical approaches is invaluable, many questions remain unanswered.
In order to better understand the biology of insulin-receptor complex, my lab will use a bioorthogonal chemistry approach to non-invasively analyze insulin-receptor binding in live animals in the context of relevant disease models.

2.2.2 Non-invasive imaging of other ligand-receptor interactions in live cells and animals
The approach described above can potentially also be used for the imaging of other cell surface ligand-receptor interactions. They might include:

  • receptor tyrosine kinases and their binding with polypeptide growth factors, cytokines, and hormones
  • G-protein coupled receptors and their interactions with multiple ligands such as hormones,nucleotides, gastrin releasing peptides, growth factors, and lipid mediators of inflammation.
  • toll-like receptors and their binding with pathogen-associated molecules such as various bacterial  cell-surface lipopolysaccharides, lipopeptides, and glycolipids.

All these receptors are involved in extracellular signal transduction and their interactions with ligands have never been imaged non-invasively on the level of the living animal. Since alterations in membrane receptor-ligand binding interactions have been linked to multiple diseases such as Alzheimer’s disease,[84] this knowledge will advance our understanding of disease biology and enable the development of improved therapeutics.

Project 3. Development of probes for image guided surgery in oncology

Introduction

Surgery still plays a central role in modern cancer treatment. A complete curative microscopic tumor resection is the single most important prognostic factor for disease-free survival in virtually all solid tumors. However, there are very few tools currently available to surgeons that would help them to achieve this important goal. The vast majority of surgeons still have to use their naked eye and tactile information to determine the extent of local invasion in surrounding tissues during the operation and the final outcome of surgery is evaluated post-operatively by immune-histopathology. As a result, even in highly expert surgical procedures, incomplete microscopic resections may lead to cancer re-growth in 40-60% of cases for several types of cancer.[85-87] Moreover, in most cases, significantly more healthy tissues and organs are removed during the surgery than it is really necessary. These compromised surgical procedures dramatically decrease the prognostic outcome of patients with cancer and significantly decrease their quality of life. Therefore, development of probes providing surgeons with clear visual feedback of cancer margins during the surgery would greatly enhance the ability to achieve complete curative treatment for many solid tumors and at the same time leave healthy tissues intact. One such probe has been recently developed for image guided surgery of ovarian cancer. [88] (http://www.nature.com/nm/journal/vaop/ncurrent/pdf/nm.2472.pdf) This technique summarized in video 1 below has been highlighted in multiple press releases.[89]

Video 1

Video 1 (courtesy of Dr. Go van Dam)

Aim 1. Development of fluorescent probes for intra-operative visualization of cancer, major blood vessels, and nerves
In close collaboration with Dr. Go van Dam, surgeon oncologist from the University Medical Center Groningen, The Netherlands (Department of Surgical Oncology), and with Prof. Vasilis Ntziachristos, who leads Institute of Biological and Medical Imaging at the Technical University of Munich, my lab will be involved in developing such multimodality probes for intra-operative fluorescent visualization of several types of cancer.  These extrinsically administered cancer-specific probes will be designed for enhancing surgical vision to outline tumor borders, tumor vasculature, metastases in the lymphatic system, locoregional metastases and possible remnant disease after surgery. In addition, we plan to develop new probes allowing real-time intra-operative visualization of major blood vessels and nerves which would greatly advance the quality of cancer surgical treatments and surgery in general.

Aim 2. Development of cancer-specific multimodality probes for diagnostic and intra-operative visualization.
Since optical imaging can be combined with other imaging modalities such as MRI/PET and CT/PET, we also plan to develop chemical probes for multi-modality imaging of cancer.  These probes would allow surgeon to operate at multiple fields of view and resolution regiments for treatment planning prior to surgery and at the same time for real-time imaging in the operating room. A single administration of a MRI/optical multi-modality or hybrid probe would enable pre-operative diagnosis and treatment planning using MRI with an option of subsequently real-time tumor dissection using the image-guided surgery approach.

For example, in esophageal cancer the number of lymph nodes affected with tumor metastases actually determines the need for curative surgery. By statistics, the surgery has not shown to be beneficial if there are more than eight lymph nodes involved. However, no existing diagnostic methods can currently identify preoperatively the actual number of affected lymph nodes. As a result, a substantial number of patients, with esophageal cancer who have 8 or more affected lymph nodes, undergo a radical surgical procedure with a high mortality and morbidity rate, without obtaining any survival benefit, but leading to decreased quality of life.

Therefore, development of multi-modality probes is crucial for many types of cancer, especially those where lymph nodes are involved, including esophageal cancer, prostate cancer, breast cancer, locally advanced rectal cancer, melanoma and peritoneal carcinomatosis of colorectal or ovarian cancer origin. Thus, my lab will also be focused on developing of multi-modality cancer specific probes based on MRI and optical imaging with the goal of direct clinical translation in close collaboration with the Department of Surgical Oncology at the University Medical Center Groningen (Dr. Go van Dam).

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  89. Accurate 5 minute background video on the full technology with Go van Dam MD, PhD http://www.licor.com/translational

ABC news video
http://abcnews.go.com/blogs/health/2011/09/19/docs-light-up-cancer-to-treat-it/

Daily Mail:
http://www.dailymail.co.uk/health/article-2038954/Glow-dark-surgery-homes-ovarian-cancer.html

HealthCanal, a very good health and science source
http://www.healthcanal.com/cancers/20901-New-imaging-technique-visualizes-cancer-during-surgery.html

Video coverage on CBS Good Morning America:
http://bit.ly/onzPao