Medium and high-throughput assays (i.e., screens) have generally not been applied to mammalian neurons because of the difficulties in culturing them in large numbers and because of the low efficiency with which the genetic makeup of neurons can be altered. Furthermore, because many aspects of neuronal function can only be assayed with electrophysiological assays, follow-up analysis and validation of screening hits is difficult. We propose to use automated imaging approaches to analyze synapse number and neuronal structure in vitro in a scalable format. We have implemented tissue culture and immunostaining approaches to monitor the number and types of synapses formed onto neurons in multi-well plates. We will couple this analysis with lentivirus mediated introduction of short-hairpin RNAs to induce RNA interference against genes expressed in neurons. This will be performed in concert with transcriptional analysis of neurons to determine the key changes in gene expression that correlate with structural and synaptic changes. The proposal represents a significant collaboration between several groups with expertise in functional analysis of neurons, automated analysis of images, viral mediated manipulation of gene expression, and whole-genome transcriptional analysis. We hope that our work will lead, for the first time, to a turn-key and robust method of analysis of neuron and synapse structure suitable for scalable, whole-genome analysis. Such a system will permit the unbiased and systematic analysis of pathways involved in neuropsychiatric diseases including neurodegenerative diseases such as Alzheimer's and Parkinson's as well as neurodevelopmental disorders such as mental retardation and autism.

Public Health Relevance

Massively parallel analysis of cells in many conditions has allowed the discovery of key pathways that control cell function. Unfortunately, these techniques have not been applied to neurons due to difficulties in handling, manipulating, and analyzing large numbers of brain cells. We propose to develop imaging-based techniques to analyze neurons in dishes at a high throughput in order to find pathways that control their development and susceptibility to disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS077907-02
Application #
8306192
Study Section
Special Emphasis Panel (ZMH1-ERB-C (05))
Program Officer
Talley, Edmund M
Project Start
2011-08-01
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
2
Fiscal Year
2012
Total Cost
$422,500
Indirect Cost
$172,500
Name
Harvard University
Department
Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Straub, Christoph; Granger, Adam J; Saulnier, Jessica L et al. (2014) CRISPR/Cas9-mediated gene knock-down in post-mitotic neurons. PLoS One 9:e105584