FY2018 Annual Report

Formation and Regulation of Neuronal Connectivity Research
Visiting Professor David Van Vactor

Abstract

The formation, refinement and maintenance of connectivity across complex neural circuits involves multiple levels of regulatory control over gene expression, from the production of mRNA to its processing, transport and translation. Our research program is focused on understanding post-transcriptional mechanisms essential for synaptic development and maintenance, and the networks of effector genes that mediate these key regulatory pathways. Using the genetic model organism Drosophila, we study transcriptome regulation within the motor system, with specific emphasis on the neuromuscular junction (NMJ) that has proven to be a powerful model for analysis of synapse function, development and disease. Our main project concerns the gene Survival of Motor Neurons (Smn), ortholog of the gene causal to Spinal Muscular Atrophy (SMA), currently thought to mediate synapse development, function and stability and protect motor neurons from degeneration and ultimate death. Through the combined application of transcriptome analysis and functional analysis in this genetic model, we are mapping the conserved mRNA targets of Smn relevant to synapse biology in order to identify key nodes and molecular pathways essential for synapse form and function.

1. Staff

Dr. David Van Vactor, Professor
Dr. Takakazu Yokokura, Group Leader (Project 1 & Drosophila Core)
Dr. Cecilia Lu, Group Leader (Project 2)
Seiko Yoshikawa, Technician (- Mar, 2019)
Thi Thu Van Dinh, Technician
Kun Xiao, Technician (- Dec, 2018)
Yu-Pei Huang, Technician (- Mar, 2019)
Rika Nakamura, Technician (Jan, 2019-)
Chie Edwards, Research Unit Administrator

2. Collaborations

2.1 Theme: Computational analysis of RNA splicing patterns in high-content RNAseq datasets, and conservation of RNA splicing and stability effects from Drosophila to human models of Spinal Muscular Atrophy (SMA).

Type of collaboration: Joint research
Researchers/Team Member:
- Dr. Margarida Gama-Carvalho (University of Lisbon, Lisbon, Portugal)
- Professor David Van Vactor
- Dr. Takakazu Yokokura
- Seiko Yoshikawa
- Thi Thu Van Dinh
- Kun Xiao

2.2 Theme: Defining patterns of RNA splicing and stability characteristic of human IPS-derived mixed motor neuron model of Spinal Muscular Atrophy (SMA).

Type of collaboration: Joint research
Researchers:
- Dr. Lee Rubin (Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA)
- Professor David Van Vactor
- Dr. Takakazu Yokokura
- Seiko Yoshikawa

2.3 • Theme: MicroRNA Modulators of Amyotrophic Lateral Sclerosis (ALS) Models in Drosophila.

Type of collaboration: Joint research
Researchers:
- Dr. Spyros Artavanis-Tsakonas (Blavatnik Institute of Cell Biology, Harvard Medical School, Boston, MA, USA)
- Professor David Van Vactor
- Dr. Takakazu Yokokura
- Seiko Yoshikawa
- Kun Xiao

3. Activities and Findings

3.1 Spinal Muscular Atrophy (SMA)

Our analysis of Smn function in regulating the formation and maintenance of connectivity in the motor circuits of the Drosophila larva reveal that this conserved gene regulates multiple aspects of presynaptic and postsynaptic structure in a way that suggests modulation of multiple trans-synaptic signaling pathways, including the Bone-Morphogenetic Protein (BMP) factor Gbb (Glass-bottom boat) and its receptors Wit (Wishful thinking) and tkv (thinkc vien), as well as fibroblast growth factor.

Over the past year, we have completed the final phase of neuronal transcriptome sequencing for the Drosophila model of SMA after optimization of the sequencing pipeline in collaboration with the OIST sequencing center. Our final RNAseq datasets are being analyzed for differential expression and exon usage in preparation for functional validation experiments in the fly model, and for comparisons to the human iPS-derived samples that are currently being sequenced. Our analysis of these data sets, through close collaboration with the labs of Drs. Gama-Carvahlo (Biosys, Lisbon) and Wall (Stanford University), will also help us determine the degree of conservation for mRNA splcing patterns across species for CNS-enriched isoforms of the genes conserved between Drosophila and human. We find hundred of genes altered in steady state level so far, and expect even higher numbers to display alterations in exon usage patterns. Current production of isoform-specific antibodies will be used to confirm the candidate Smn effectors in several key pathways, as a prelude to isoform-specific transgenic rescue experiments.

4. Publications

4.1 Journals

Nothing to report

4.2 Books and other one-time publications

Lu, C.S. and Van Vactor, D. (2019) Genetic Analysis of Synaptogenesis in Cellular Migration and Formation of Neuronal Connections: Comprehensive Developmental Neuroscience, Volume 2. Elsevier Ltd., London, UK.

4.3 Oral and Poster Presentations

Yokokura, T., Yoshikawa, S., Chobanyan, T., Goto, H., AF dos Santos, H., Fonseca, A., Lynes, M., Chang, H., Dimlich, D., Kankel, M., Sen, A., Artavanis-Tsakonas, S., Rubin, L., Gama Carvalho, M, Van Vactor, D. mRNAs Altered in Human and Drosophila Models of Spinal Muscular Atrophy. Workshop on Integrative Approaches in Neurodegeneration; Lisbon, Portugal. June 21-23, 2018
Yokokura, T., McNeill, E.M., Chobanyan, T., Yoshikawa, S., Goto, H., AF dos Santos, H., Fonseca, A., Lynes, M., Rubin, L., Gama Carvalho, M, Van Vactor, D. Mutants Lacking Drosophila Smn Display SMA Disease-relevant Neuromuscular Defects that can be Rescued by Elevation in Conserved Transynaptic Signaling Pathway. Workshop on Integrative Approaches in Neurodegeneration; Lisbon, Portugal. June 21-23, 2018
Yokokura, T., AF dos Santos, H., Yoshikawa, S., Fonseca, A., Lynes, M., Rubin, L. L., Gama Carvalho, M., Van Vactor, D. Defining Conserved Gene Networks on SMN function using Drosophila SMA model and human iPS driven neural cells. 22nd International SMA Researcher Conference; Dallas TX, USA. June 14-16, 2018
Van Vactor, D., AF dos Santos, H., Yoshikawa, S., Fonseca, A., Lynes, M., Rubin, L. L., Gama Carvalho, Yokokura, T. (2018): JPND Workshop on “Integrative Approaches in Neurodegeneration”, U. Lisboa, Lisbon, Portugal.
Van Vactor, D., AF dos Santos, H., Yoshikawa, S., Fonseca, A., Lynes, M., Rubin, L. L., Gama Carvalho, Yokokura, T. (2019): ”Modeling Degenerative Disease in the Drosophila Motor System” HU MARC U-START Program, Hampton University, Hampton, VA.
Van Vactor, D., AF dos Santos, H., Yoshikawa, S., Fonseca, A., Lynes, M., Rubin, L. L., Gama Carvalho, Yokokura, T. (2019) ”Modeling Degenerative Disease in the Drosophila Motor System” [Keynote lecture] Annual Symposium, Developmental Biology Training Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.
Van Vactor, D., AF dos Santos, H., Yoshikawa, S., Fonseca, A., Lynes, M., Rubin, L. L., Gama Carvalho, Yokokura, T. (2019): ”Modeling Spinal Muscular Atrophy in the Drosophila Motor System” HMS Initiative for RNA Medicine Seminar Series, Beth Israel Deaconess Medical Center Cancer Center, Boston, MA.

5. Intellectual Property Rights and Other Specific Achievements