Hwai-Jong Cheng, MD, PhD - US grants

DESCRIPTION (provided by applicant): The nervous system consists of an intricate network of neuronal connections. The formation of proper connections during development is the essential first step in building a functional nervous system. The overall goal of this study is to understand the molecular mechanisms that guide axons to find their appropriate targets. Gene targeting techniques, in vitro molecular binding experiments, and in vitro neuronal cultures will be used to study specific functional role of axon guidance molecules. The specific aims of this proposal are: (1) to study the specific functions of plexins by analyzing the axon guidance phenotypes in plexin knockout mice, (2) to characterize the interactions of plexins by analyzing the phenotypes in double knockout mice and by studying the in vitro biochemical interactions, and (3) to explore the role of plexins in stereotyped axon pruning by analyzing the pruning phenotypes in plexin knockout mice and by establishing in vitro axonal branch pruning assays. This research will help us understand how axon guidance molecules function together to ensure that during the development of the nervous system no errors occur in the formation of the initial neuronal network. More generally, defects in forming appropriate axonal connections are likely to cause many neurodevelopmental disorders, such as autism, schizophrenia, synesthesia and mental retardation. Understanding the molecular mechanisms could revolutionize our ability to characterize and treat these developmental disorders.

DESCRIPTION (provided by applicant): Degenerative diseases that result in the loss of photoreceptors, including Age-related Macular Degeneration (AMD) and Retinitis Pigmentosa (RP), are common causes of blindness in the developed world. These diseases largely spare the connections and functions of the retinal ganglion cells that link the retina to central targets in the brain. Therefore, the strategy of rendering retinal ganglion cells directly responsive to light holds a great deal of promise for developing therapies for these diseases. Light-sensitive ganglion cells would bypass the damaged photoreceptors, but take advantage of the normal visual pathways beyond the retina. Other laboratories have previously demonstrated that it is possible to express the light-activated cation channel, channelrhodopsin-2 (ChR2), in retinal cells. Studies have shown that in a rodent model of photoreceptor degeneration, expression of ChR2 can render retinal cells photosensitive. Light responses due to the expression of ChR2 in the retina are transmitted to the visual cortex, and can underlie rudimentary visual behaviors. A great deal of work remains to be done, however, before the use of ChR2 or similar light- activated molecules to treat retinal degenerative diseases could be contemplated in a clinical setting. This R21 proposal addresses two important issues that need to be tackled. Specific aim 1 will determine how best to obtain widespread, stable expression of ChR2 in retinal ganglion cells in a non- rodent model system, and whether such expression leads to visual responses in retinal ganglion cells that are transmitted to lateral geniculate nucleus and primary visual cortex cells. Specific aim 2 will determine whether the vision caused by the expression of ChR2 in retinal ganglion cells can either enhance or interfere with the development and adult function of normal visual pathways. Successful completion of these two aims will bring the field significantly closer to the goal of being able to translate light-activated channel technology into the clinic, and will provide preliminary data needed to apply for funding to determine whether this approach can be used to produce behaviorally useful vision in a retinal detachment model of blindness. PUBLIC HEALTH RELEVANCE: The long-term goal of this research is to develop methods to cure forms of blindness caused by the loss of photoreceptor function including Age-related Macular Degeneration and Retinitis Pigmentosa. Experiments will test whether artificially making retinal cells photosensitive through expression of light- activated ion channels is a promising approach to curing these diseases.

DESCRIPTION (provided by applicant): In the mature mammalian visual system, information from the two eyes is anatomically segregated into layers in the lateral geniculate nucleus, and into ocular dominance columns in primary visual cortex. During the past half century, the development of this segregation has served as a premier model for the development of specific connections in the brain. Groundbreaking work in the 1960s showed that neuronal activity is important for the normal development of ocular dominance columns. If poor vision or no vision is experienced by one eye early in life, then that eye loses anatomical and functional connections to the brain. This results in blindness through the affected eye later in life, even if the cause of the poor vision is fixed so that the eye itself functions normally. Many studies over the ensuing years have looked at the effects of altering or abolishing neuronal activity on the development of eye-specific segregation. Findings from these studies lead to the following conclusions: 1) During development, specific connections in the visual system emerge over time from initially diffuse connections, 2) abolishing retinal activity during development maintains the initial imprecise connections, and 3) temporal correlation in the firing patterns of neurons within each eye, and lack of correlation between eyes is responsible for the development of eye-specific connections. These conclusions formed the dogma of the field of development of sensory systems, and significantly influenced related fields such as neurodevelopmental disorders, adult plasticity in the brain, and learning and memory. However, new experiments over the past decade have questioned each of these conclusions. This proposal will address the controversies by performing the following specific aims. 1) What is the normal pattern of development of ocular dominance columns? Anatomical tracing methods will be used to determine whether afferents serving the two eyes are initially overlapping or segregated in primary visual cortex. 2) What is the effect of complete retinal activity blockade on the development of eye-specific segregation in the LGN and cortex? Pharmacological agents will be used to silence retinal ganglion cell action potentials during development, and the effects on segregation will be examined. 3) What aspects of retinal ganglion cell activity are important for normal development of eye-specificity in the LGN? Manipulations that alter patterns of activity in the retina will be compared in order to determine which aspects of activity patterns are associated with normal segregation versus lack of segregation. 4) What is the role of correlated activity in the development of segregation? Light activatable molecules will be used to control activity patterns to determine whether increased correlation of firing of neighboring cells between the two eyes leads to lack of eye-specific segregation. These experiments will not only help to resolve important basic science controversies, but will also have clinical implications. Understanding the initial state of connections in the brain, the effects of absent or altered activity, and the key aspects of activity patterns that are needed for normal development, will all aid in the understanding and eventual treatment of neurodevelopmental disorders.

The birth of new neurons (called neurogenesis) in the adult hippocampus is critical for learning and memory, and disruption of this process during aging is associated with neuropsychiatric illnesses that undermine cognition in the aged brain. Most of our knowledge about adult neurogenesis relates to the survival and differentiation of newborn neurons in the young adult brain. Much less is known about how these neurons integrate into existing neural circuits in the aged hippocampus. Neuronal progenitors in the hippocampus give rise to granule cells that, when fully differentiated, send axons along the mossy fiber pathway, where they form synaptic connections (called boutons) to CA3 pyramidal neurons. Previously we developed a serial immuno- electron microscopic approach to study the development of these newborn mossy fiber boutons in the adult brain. Here, using a reporter mouse that we can induce to label the new neurons that are born in a particular time period, we investigate the development and integration of newborn granule cells in the aged hippocampus. This mouse line allows us to birth-date and characterize neurogenesis at any age, including in aged mice 18 months or older. Our preliminary studies show that the progenitor pool changes in the aged hippocampus; more quiescent (inactive) progenitors are present compared to young-adult brain. We have also found the potential for newborn granule cells to form de novo synapses in aged brain is significantly reduced; instead existing boutons have to be replaced when these newborn neurons form synapses. These results reveal previously unknown changes in newborn neurons and their progenitors in the aged brain. In this proposal, we focus on three questions. (1) What are the molecular phenotype and developmental origin of the neuronal progenitors in aged hippocampus? These experiments will reveal how progenitors in the aged brain are different from those in young adults. (2) What is the age and developmental origin of the existing boutons that are replaced by the newborn mossy-fiber boutons in aged brain? Why do the newborn granular cells in the aged brain lose their ability to form de novo synapses? Is this loss due to changes in the neuronal progenitors or to changes to the environment in the aged hippocampus? The answers to these questions will help us understand the specific functional role of adult neurogenesis in the aged brain. (3) How do changes in neuronal activity affect neurogenesis in the aged brain? We have found that the aged hippocampus loses a voltage- gated potassium channel that regulates neuronal intrinsic excitability, and that this channel has a significant effect on adult neurogenesis. We will ask how the changes in neuronal activity resulting from the loss of this channel affect the development and integration of newborn granule cells in the aged hippocampus. These experiments will fill an important gap in our knowledge about neurogenesis in the aged brain, which we expect will contribute to the future development of rehabilitative or therapeutic strategies to improve the function of the aging brain.

The birth of new neurons (called neurogenesis) in the adult hippocampus is critical for learning and memory and disruption of this process is associated with human neurological disorders such as Alzheimer?s disease. Rates of adult hippocampal neurogenesis (AHN) are tightly linked with changes in physiological activity. Activities such as enhanced exercise or learning as well as pathophysiological changes such as epilepsy, profoundly alter AHN. Knowing how ANH neurogenesis regulates neuronal circuitry is therefore important for understanding its overall impact on brain physiology. Central to this issue is understanding how newborn neurons in adult brain achieve long-term integration. Understand the molecular and cellular mechanisms regulating synaptic integration in AHN could lead to selective pharmacological targets for functional improvement during pathological conditions or in aging where levels of adult neurogenesis are dramatically decreased. Neuronal progenitors in the adult hippocampal dentate gyrus give rise to newborn granule (GCs) cells that, when fully differentiated, receive synaptic inputs from entorhinal cortex and send axons along the mossy fiber pathway to form synaptic outputs with CA3 pyramidal neurons. We and others have shown that it takes about eight weeks for the newborn GCs to fully differentiate and form mature synaptic inputs and outputs. The major focus of this proposal is to determine the molecular changes in the synaptic outputs when newborn mossy fiber boutons from GC are forming synapses with mature CA3 pyramidal cells. We propose to use superresolution immunofluorescent array tomography and conjugate array tomography coupled with electron microscopy to profile the proteomic changes of the pre- and post-synaptic elements during the establishment of mature synapses. We have found that to establish a mature synaptic contact the mossy fiber can either 1) form a de novo nascent synapse or 2) replace an existing mossy fiber bouton assuming control of the existing postsynaptic CA3 dendrite. The molecular mechanisms regulating these disparate cellular processes are unknown. Here we will use array tomography analysis to profile the proteomic changes in these synapses to test the hypothesis that the synaptic molecular composition of integrating newborn neurons in adult hippocampus is highly dynamic during the entire maturation process. We have two main focuses: (1) to establish a proteomic profile of the developing and mature presynaptic mossy fiber terminal during adult hippocampal neurogenesis and (2) to establish a proteomic profile of the developing and mature postsynaptic mossy fiber terminal during adult hippocampal neurogenesis. These experiments will be the first to address the intricate proteomic changes essential for establishing new synaptic outputs during adult neurogenesis and will potentially identify a pharmacological target for therapeutic strategies to improve the function of adult brain.