The main goal of this proposal is to generate new bioinformatics tools and mouse transgenic reagents for monitoring astrocyte gene expression in the striatum and amygdala. These experiments are designed to: (1) reveal how transplanted human astrocytes react to engraftment, (2) reveal how transplanted astrocytes influence their surroundings in mouse Rett models (MeCP2 mutant) and anxiety models (SERT mutant), and (3) to create novel transgenic animals for characterization of astrocyte heterogeneity and function. The existing tools and methods to address these questions are inadequate, in part because the dynamic range and sensitivity of microarrays is limited, FAC-sorting and immunopanning risk causing cellular changes during the purification, and laser capture requires massive RNA amplification.
Two Aims will develop new methods and reagents to address these limitations.
In Aim 1 a bioinformatics pipeline will be optimized to identify the origin of transcipts from mouse and human cells. Next, human astrocyte progenitors derived from H9 ES cells or Qthera human fetal glial-restricted precursors (GRPs, "Q cells") will be stereotaxically transplanted into the striatum and amygdala of mice and allowed to mature. High content long-read RNA-sequencing and bioinformatics will be used to deconvolute the origin of transcripts from the human-into-mouse transplants. This species-specific gene profiling data should provide new information on how human astrocytes precursors behave in the striatum versus the amygdala. At the same time the response of the striatum and amygdala to the transplanted cells will be monitored. Finally, how astrocytes respond-to and influence the striatum and amygdala of MeCP2 and SERT mutant mice will be examined, using RNA-sequencing. Transplantation of astrocyte precursors is being considered for many clinical trials to treat mental illness, disease and CNS injury. These bioinformatics methods and new data sets should be useful for further characterization of astrocyte function.
In Aim 2 novel mouse lines will be generated to enable the selective purification of RNAs from astrocyte-subtypes and lineally related neurons present in heterogeneous brain tissue. Transgenic mice will be created that express the ribosomal affinity tag Rpl22 and the fluorescent calcium reporter GCaMP3 under the control of the astrocyte promoter GFAP or the neuron promoter Synapsin. These reporters will be restricted to glial and neuronal subtypes using an intersectional approach that requires Cre-recombination. These transgenics will be characterized using crosses to progenitor-specific and inducible Cre lines to activate the reporters in subsets of glia. RNAs bound to Rpl22 will be isolated and sequenced to determine whether glia and neurons originating from the same progenitor cells share molecular-genetic features. These new mouse lines should have broad value to the neuroscience community and help to identify molecular features that either bind or distinguish subsets of astroctyes and neurons arising from shared progenitors.

Public Health Relevance

Astrocytes are abundant in the brain, but represent a poorly understood cell population that is important for understanding mental illness and neurodegeneration/regeneration. This proposal will develop new bioinformatic methods and transgenic mouse lines to monitor the genetic profile of astrocyte subtypes. Our studies are designed to provide new information about the heterogeneous characteristics of astrocytes, their ability to differentiate, integrate and function when transplanted, and to identify the interactions that occur between astrocytes and their surrounding cells within the brains of mice with mutations causing autistic- (MeCP2 mutant) and anxiety-related (SERT mutant) behaviors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Project (R01)
Project #
1R01MH099571-01
Application #
8442761
Study Section
Special Emphasis Panel (ZMH1-ERB-M (06))
Program Officer
Beckel-Mitchener, Andrea C
Project Start
2013-01-01
Project End
2016-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
1
Fiscal Year
2013
Total Cost
$421,692
Indirect Cost
$142,668
Name
Salk Institute for Biological Studies
Department
Type
DUNS #
078731668
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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