Bonnie L. Firestein - US grants

The brain is an incredibly complex organ. If neurons do not talk to their partners, the brain does not function. How does the brain manage to allow the correct communication to occur? One way is to regulate the amount of signaling proteins that are used by the neurons. The purpose of the proposed research is to understand how the brain performs this process. Dr. Firestein has cloned a protein called cypin which is involved in making sure that there is a correct amount of proteins at sites where neurons communicate. The proposed studies will first investigate how cypin performs this function by analyzing portions of cypin and determining which regions are responsible for cypin function. Second, Dr. Firestein will analyze why cypin regulates the localization of some proteins better than others. Finally, Dr. Firestein will determine what happens when cypin levels are increased in neurons. The results of these studies will increase our understanding of basic mechanisms that underlie brain development. In addition, the performance of these experiments will be part of a program to train undergraduate and graduate students in cutting edge-techniques in neuroscience. Two doctoral students, one of whom is an underrepresented minority, and two undergraduate students, will be involved in performing the experiments proposed. In addition, Dr. Firestein will be developing an Advanced Neurobiology Laboratory to prepare undergraduate students for bench work in laboratories at Rutgers University. Taken together, the proposed project will help us to understand how the brain is wired for communication, at the same time providing opportunities for students to conduct bench science.

Firestein - Identification of Core Pathways that Regulate Dendrite Morphology

Proposal 0548543

Abstract

In order for proper neuronal function to occur, the neuron must have the correct number of input centers, or dendrites, which look like branches on a tree. However, very little is currently known about how the pattern of these branches is determined. The goal of Dr. Firestein's work is to identify core pathways by which dendrite number is regulated. Dr. Firestein will make use of molecular biology, biochemistry, and neuronal cell culture to investigate these pathways. First, Dr. Firestein will establish four criteria by which to characterize regulators of dendrite number. This system, not unlike criteria used to characterize neurotransmitters, will help to identify whether proteins reported in the literature are indeed members of core pathways for dendrite branching. Experiments will focus on two proteins, cypin and PSD-95, which Dr. Firestein believes to be components of the core pathway. Since the cytoskeleton, or supporting structure of a dendrite, must change when a dendrite branches, experiments will assess whether cypin and PSD-95 change dendrite number by altering the cytoskeleton. Second, the proposed studies will be extended to assess the role of other presumed proteins in the core program of dendrite branching. Other proteins will be tested to see if they they act together with cypin and PSD-95 by using cultured hippocampal neurons that have altered levels of these proteins. Finally, Dr. Firestein's group will take images of cultured hippocampal neurons over time using both static pictures and video microscopy and identify how dendrites form: via outgrowth, sprouting, or retraction. These studies are important because they will provide information on how dendrite branching is regulated during development to yield a functional brain. The impact of this research is far-reaching.

The performance of the experiments will be part of a program to train undergraduate and graduate students in cutting-edge techniques in neuroscience and in practical aspects of experimental design and data interpretation. Furthermore, Dr. Firestein continues to mentor teachers on sabbatical from high school. By exposing high school teachers and students to experimental biology, the proposed research will motivate future scientists at a very early stage in their careers.

For proper neuronal function to occur, the neuron must have the correct number of input centers, or dendrites, which look like branches on a tree. However, very little is currently known about how the pattern of these branches is determined. The goal of this project is to identify how both global and local changes in dendrite branching occurs. The Principal Investigator and her students will make use of molecular biology, biochemistry, and neuronal cell culture to study signaling by brain-derived neurotrophic factor (BDNF), a major regulator of dendrite branching. First, they will assess how administration of BDNF to neurons changes the shape of the dendrites overall and at distinct regions from the cell body. Second, neurons will be exposed to BDNF-coated latex beads to mimic local stimulation, and changes in the dendrite branching by this treatment will be compared to those that occur with global BDNF treatment. Third, they will use drugs and molecular techniques to identify the proteins that are responsible for the effects of BDNF. These studies are important because they will provide information on how dendrite branching is regulated during development to yield a functional brain. The impact of this research is far-reaching. Conducting the experiments will not only advance our understanding of how dendrite morphology is regulated but will also be part of a program to train high school, undergraduate, and graduate students in cutting-edge techniques in neuroscience. The project involves setting up an exchange program for undergraduate and Masters students at the University of Liberia so that they can perform summer research in the Firestein laboratory. A major goal is to establish an international program to bring neuroscience to the University of Liberia community through seminars, workshops, and exchange programs. This program will help educate people in a country facing global conflict.

For proper neuron function, the neuron must have the correct number of input dendrites, which look like branches on a tree. Very little is currently known about how the pattern of these branches is determined or how these branches change when a person learns. To make new dendrite branches, a cytoskeleton must be produced, much like the skeleton in fingers. Dr. Firestein discovered a protein called cypin that plays a critical role in making this cytoskeleton, and hence dendrites. It is hypothesized that without cypin, neurons will not form properly, and the brain will not function correctly. Using nerve cells in a dish, undergraduate and graduate students will perform experiments to understand the mechanism by which cypin acts to yield functioning neurons. By investigating how cypin gives nerve cells their shape and how these nerve cells integrate into simple circuits, this research will aid in our understanding of principles by which circuits may be modified during events, such as learning. Dr. Firestein and colleagues will include a diverse group of undergraduate and graduate students and will establish an exchange program with University of Puerto Rico. This proposal also encompasses activities to excite a younger generation of students (K-12) about neuroscience by producing a video series "Teach Me Neuroscience" and training K-12 teachers at the bench. It is Dr. Firestein's hope to establish a program to bring neuroscience to the community in Puerto Rico via seminars, workshops, and exchange programs.

The specific goal of the current work is to evaluate how cypin and its binding partner PSD-95 affect neural circuit dynamics. Experiments aim to determine the mechanism by which cypin decreases synaptic PSD-95 using viral-mediated gene expression in cell culture to alter cypin levels. The role of the proteasome in cypin-mediated changes in PSD-95 will be assessed. It will also be determined whether cypin and PSD-95 levels affect function and activity of neural circuits in vitro. Dr. Firestein has a comprehensive set of tools available to manipulate cypin functionality at the molecular level and will assess effects of altered cypin levels on dendrite number, spine number, and size. Experiments will make use of electrophysiology techniques to determine whether cypin affects neuronal signaling. The proposed work uses interdisciplinary approaches - molecular/cellular, biochemical, and electrophysiological - to understand how morphological changes to neurons result in changes in synaptic function, making a large advance from previous cell culture work and defining a mechanism by which cypin acts to regulate dendritogenesis and determine effects on neural circuitry.

PI: Yarmush, Martin L.
Proposal Number: 1512170

Traumatic brain injuries (TBI) are the leading cause of disability each year in the US and are also a major risk factor for epilepsy in both injured civilian and military populations. TBI dramatically reduces quality of life in affected patients and there are significant direct and indirect costs associated with TBI. While some drug TBI treatment protocols are under clinical review, none has been identified which can significantly attenuate the progression of events leading to neurological impairment. Improved in vitro screening methods are critical to expedite drug identification and development. Animal studies are both expensive and time consuming, but most in vitro approaches fail to recapitulate in vivo central nervous system inter-cellular connections and responses. Therefore, the goal of the proposed studies is to develop a novel high content "Brain-on-a-Chip" device, which integrates pairs of brain tissue slices and uses novel microfabrication and optical imaging tools, to identify drug candidates that can be used to treat TBI.

Many recent studies indicate that mitochondrial dysfunction contributes to secondary TBI severity and associated axonal dysfunction. As such, the investigators aim to develop a high-content approach to screen mitochondrial drugs to alleviate post-TBI neuronal decay. An interdisciplinary team of science and engineering investigators will utilize microfabrication techniques to develop a "Brain-on-a-Chip" device which will be used to culture paired brain organotypic tissue slices with individual interconnecting axons that extend over microchannels. Strain injury will be introduced by pressurizing a cavity beneath the microchannels. Integrating a multi-electrode array (MEA) on-chip will enable precise and on-line identification of electrophysiological changes in response to injury. The investigators expect to assess how various strain injuries affect electrophysiological and biochemical responses between two organotypic slices using a novel dynamic optical imaging approach. By using microfabricated "Brain-on-a-Chip" arrays, the investigators will be able to screen, in parallel, drug candidates both individually and in combination, more efficiently than has been previously possible. Establishment of such a novel platform is significant, because it would accelerate the identification of molecular entities which control the injury response and, in concert, the development and screening of drug treatments for complex circuit disorders like TBI and epilepsy. The education plan includes high school, undergraduate, and graduate training components with a focus on underrepresented student education. Furthermore, industrial practitioners will be involved in bioengineering courses, which is an effective approach allowing student exposure to the industrial environment.

Neurons, the basic cells of nerves, are one of the most difficult to regenerate after disease or injury. If advances are going to be made in engineering systems that support nerve regrowth and regeneration, it is imperative that we develop a full understanding of how these cells respond to their biomechanical environment. This study will investigate how changes in the extracellular mechanical environment affects the development of neurons and the branching of dendrite -- the structure that receives signals from other nerve cells. Knowledge gained from this project will help to develop strategies that may eventually improve treatments for degenerative neural diseases or following nerve injury due to trauma. The research team will integrate students at all levels, from graduate school through high school, as well as teachers into the project. In addition, outreach will involve the development of hands-on projects and demonstrations that can introduce the public to concepts of neuroscience, imaging, and biomechanics.

Two hypotheses are being investigated related to neuron growth and development within this project. The first is that local structural changes associated with applied cell forces act to transduce extracellular mechanical cues to the cytoskeleton. The second is that local changes in cellular tension in response to changes in the extracellular mechanical microenvironment act to transduce extracellular mechanical cues to the cytoskeleton. The research team will make use of a vinculin force sensor for which the tension can be measured using Forster resonance energy transfer (FRET). This mechanical measurement will be combined with fluorescence imaging of the cytoskeletal dynamics. Thus, the specific aims of this project are to: 1) demonstrate that vinculin expression changes as a function of substrate stiffness and tension; 2) demonstrate that vinculin tension is altered in response to changes in substrate stiffness and investigate the role of integrin, cadherin, and neurotransmitter receptors in mediating this response; and 3) investigate the relationship between vinculin tension, microtubule assembly, and the resulting dendritic branching.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

PROJECT SUMMARY Dendrite morphology determines many aspects of neuronal function, including action potential propagation and information processing. Although emerging evidence suggests that dendrite growth and branching is regulated locally, the lack of optimal measurements at local sites exists. Brain-derived neurotrophic factor (BDNF) is one of the most studied regulators of dendrite development, and we reported that BDNF exerts distinct local effects on the dendritic arbor depending on where on the arbor it is applied. BDNF triggers PKA activation to regulate dendrite branching, yet not much is known about how BDNF activates PKA to promote local dendrite branching. The proposed work aims to utilize cAMP-dependent protein kinase (PKA) activation sensors as part of a F?rster resonance energy transfer (FRET)-based imaging platform to study the spatiotemporal regulation of the dendritic arbor by PKA activation. First, we will locally apply PKA activator or inhibitor to the dendrite and show a functional relationship between PKA activity and dendrite branching. A microtubule targeted A-kinase activity reporter (tAKAR4?) that shows high sensitivity and dynamic range and is activated by neuromodulators will be expressed in cultured embryonic rat hippocampal neurons of both sexes. We will measure the time-dependent dynamic distribution of PKA in neurons as dendrites develop and branch over time. Second, it has been reported that nuclear signaling of activated PKA occurs and is highest after stimulation of secondary versus other order dendrites, regardless of distance from the soma. As such, we will construct a new PKA activation sensor, tAKAR4?, that will be targeted to the nucleus and use this new FRET sensor to determine whether nuclear PKA activity increases when secondary, but not other order, dendrites are stimulated with PKA activator. Third, we will use the tAKAR4? FRET sensor and nuclear- targeted AKAR4 probe to determine the mechanism by which BDNF locally regulates the arbor. These studies will shed light on mechanisms that shape neuronal morphology that can then be targeted for therapeutics to restore neuronal connectivity and circuitry after injury due to stroke or TBI or as a result of neurodegenerative diseases.