Kevin M. Wright - US grants

DESCRIPTION (provided by applicant): Cell death by apoptosis is triggered when cytochrome c is released from the mitochondria to activate the caspase proteases. Our results indicate that mitotic cells and postmitotic neurons regulate cytochrome c-mediated caspase activation in fundamentally different ways. Whereas cytochrome c is sufficient to induce rapid caspase activation and apoptosis in many mitotic cells including naive PC 12 cells, the neuronally-differentiated PC12 cells and primary sympathetic neurons are remarkably resistant to cytochrome c due to strict regulation by the inhibitor of apoptosis proteins (lAPs). Our preliminary data indicate that this differential regulation of apoptosis is not because of any differences in lAPs themselves in mitotic cells and postmitotic neurons, but because of differences in the rate of caspase activation controlled by Apaf-1. Our hypothesis is that a decrease in Apaf-1 function with neuronal differentiation is critical for engaging lAPs to strictly regulate apoptosis in neurons. The aims of this proposal are to test this hypothesis in the model of differentiating PC12 cells and primary neurons with a specific focus on examining how transcriptional and posttranslational changes in Apaf-1 with neuronal differentiation control the selective, strict regulation of apoptosis by lAPs in neurons.

Project Summary The formation of functional neural circuits requires the proper migration of newly generated neurons, precise guidance of axons to the appropriate postsynaptic targets, the formation of elaborate dendritic arbors and establishment of synaptic connections. These processes depend on the reiterative use of cell-surface receptors throughout development that respond to instructive cues present in the extracellular environment. The transmembrane glycoprotein dystroglycan is involved in multiple aspects of neural circuit development, including axon guidance and dendritic arborization. The objective of this proposal is to define the specific molecular pathways that dystroglycan functions within to regulate these processes. The first aim tests the hypothesis that dystroglycan interacts with the planar cell polarity (PCP) protein Celsr3 to regulate axon guidance. The experiments proposed in this aim will define which cellular populations dystroglycan functions in to regulate axon guidance in vivo, determine how dystroglycan and Celsr3 interact in vitro and in vivo and investigate how the loss of dystroglycan affects signaling downstream of PCP pathway activation. The second aim will focus on understanding the role of dystroglycan in regulating dendritic arborization. Specifically, the experiments outlined will examine how dendritic morphology is affected in models of dystroglycanopathy, determine if dystroglycan functions cell-autonomously and provide mechanistic insight into the intracellular signaling cascades downstream of dystroglycan that regulate dendritic morphology. Taken together, the proposed experiments will define the role of dystroglycan in regulating multiple aspects of neural circuit development and provide insight into how these processes are affected in individuals with mutations that affect normal dystroglycan function.

PROJECT SUMMARY/ABSTRACT Amacrine cells (ACs) are the principle inhibitory neurons of the inner retina, with at least 45 morphologically distinct subtypes that can be distinguished by the size, shape, and stratification of their dendritic arbors within the inner plexiform layer (IPL). Most ACs do not have axons, and instead integrate signaling across their dendritic arbors. This unique arrangement means that form and function are intimately related in ACs, perhaps more so than in any other neuron type. However, we know virtually nothing about how individual AC subtypes are specified during development, or how their adopt their stereotyped morphologies. We hypothesize that selectively expressed transcription factors (TFs) are well poised to direct subtype-specific genetic programs that specify and direct the morphological maturation of ACs. Within the AC population, the homeodomain TF Isl1 is only expressed in starburst amacrine cells (SACs) and Gbx2 is only expressed in a previously unidentified population of medium-field non-GABAeric, non-Glycinergic ACs (MF-nGnGs). Our preliminary results show that early deletion of Isl1 or Gbx2 from AC precursors results in defects in SAC and MF-nGnG subtype specification, respectively. Later deletion of Isl1 or Gbx2 in post-migratory SACs or MF-nGnGs alters their dendritic morphology and stratification patterns. Therefore, Isl1 and Gbx2 appear to be required for the initial specification and the subsequent morphological maturation of their respective AC subtypes. We will test this in the following Specific Aims: 1) Determine whether Isl1 and Gbx2 are necessary and sufficient for AC subtype specification and maturation. We will use conditional loss- and gain-of function approaches to determine whether Isl1 and Gbx2 are both necessary and sufficient to initiate and maintain terminal differentiation in SACs and MF-nGnGs, respectively. Furthermore, we will use a combination of genomic approaches (RNASeq, ATACseq) to identify the Isl1 and Gbx2 gene regulator networks in the respective AC subtypes. 2) Define how Isl1 and Gbx2 regulate SAC and MF-nGnG morphology and functional connectivity. We will first identify how the loss of Isl1 in SACs and Gbx2 in MF-nGnGs affects the development of their stereotyped dendritic morphology and stratification patterns. Then we will test candidate effector genes and pathways for their involvement in the establishment of dendritic arbors. Finally, we will determine how the loss of Isl1 and Gbx2 affects the functional connectivity of SACs and MF-nGnGs in visual circuits. Together, these experiments are expected to reveal how selectively expressed transcription factors specify AC subtypes and drive terminal differentiation programs that endow ACs with their unique morphological properties. We expect that these findings will reveal principles that are broadly applicable across the developing nervous system.

PROJECT SUMMARY/ABSTRACT Amacrine cells (ACs) are the principle inhibitory neurons of the inner retina, with at least 45 morphologically distinct subtypes that can be distinguished by the size, shape, and stratification of their dendritic arbors within the inner plexiform layer (IPL). Most ACs do not have axons, and instead integrate signaling across their dendritic arbors. This unique arrangement means that form and function are intimately related in ACs, perhaps more so than in any other neuron type. However, we know virtually nothing about how individual AC subtypes are specified during development, or how their adopt their stereotyped morphologies. We hypothesize that selectively expressed transcription factors (TFs) are well poised to direct subtype-specific genetic programs that specify and direct the morphological maturation of ACs. Within the AC population, the homeodomain TF Isl1 is only expressed in starburst amacrine cells (SACs) and Gbx2 is only expressed in a previously unidentified population of medium-field non-GABAeric, non-Glycinergic ACs (MF-nGnGs). Our preliminary results show that early deletion of Isl1 or Gbx2 from AC precursors results in defects in SAC and MF-nGnG subtype specification, respectively. Later deletion of Isl1 or Gbx2 in post-migratory SACs or MF-nGnGs alters their dendritic morphology and stratification patterns. Therefore, Isl1 and Gbx2 appear to be required for the initial specification and the subsequent morphological maturation of their respective AC subtypes. We will test this in the following Specific Aims: 1) Determine whether Isl1 and Gbx2 are necessary and sufficient for AC subtype specification and maturation. We will use conditional loss- and gain-of function approaches to determine whether Isl1 and Gbx2 are both necessary and sufficient to initiate and maintain terminal differentiation in SACs and MF-nGnGs, respectively. Furthermore, we will use a combination of genomic approaches (RNASeq, ATACseq) to identify the Isl1 and Gbx2 gene regulator networks in the respective AC subtypes. 2) Define how Isl1 and Gbx2 regulate SAC and MF-nGnG morphology and functional connectivity. We will first identify how the loss of Isl1 in SACs and Gbx2 in MF-nGnGs affects the development of their stereotyped dendritic morphology and stratification patterns. Then we will test candidate effector genes and pathways for their involvement in the establishment of dendritic arbors. Finally, we will determine how the loss of Isl1 and Gbx2 affects the functional connectivity of SACs and MF-nGnGs in visual circuits. Together, these experiments are expected to reveal how selectively expressed transcription factors specify AC subtypes and drive terminal differentiation programs that endow ACs with their unique morphological properties. We expect that these findings will reveal principles that are broadly applicable across the developing nervous system.